DNA strands useful for the synthesis of xanthophylls and the process for producing the xanthophylls

ABSTRACT

Disclosed are the following DNA strands relating to the synthesis of keto group-containing xanthophylls such as astaxanthin and the like, and the techniques relating to the production of xanthophylls by genetic engineering: 
     A DNA strand having a nucleotide sequence which encodes a polypeptide having an enzyme activity for converting a methylene group at the 4-position of β-ionone ring into a keto group. 
     A DNA strand having a nucleotide sequence which encodes a polypeptide having an enzyme activity for converting a methylene group at the 4-position of a 3-hydroxy-β-ionone ring into a keto group. 
     A DNA strand having a nucleotide sequence which encodes a polypeptide having an enzyme activity for adding a hydroxyl group to the 3-carbon of a 4-keto-β-ionone ring. 
     It is possible to produce a variety of xanthophylls such as canthaxanthin, astaxanthin and the like by introducing the DNA strands into an appropriate microorganism such as Escherichia coli and the like.

TECHNICAL FIELD

The present invention relates to DNA strands useful for the synthesis ofketo group-containing xanthophylls (ketocarotenoids) such as astaxanthinwhich are useful for heightening the color of cultured fishes andshellfishes such as sea breams, salmons, lobster and the like and isused for foods as a coloring agent and an antioxidant, and to a processfor producing keto group-containing xanthophylls (ketocarotinoids) suchas astaxanthin with use of a microorganism into which the DNA strandshave been introduced.

BACKGROUND ART

The term xanthophylls mean carotenoid pigments having anoxygen-containing group such as a hydroxyl group, a keto group or anepoxy group. Carotenoids are synthesized by the isoprenoid biosyntheticprocess which is used in common halfway with steroids and otherterpenoids with mevalonic acid as a starting material. C15 farnesylpyrophosphate (FPP) resulting from isoprene basic biosynthetic pathwayis condensed with C5 isopentenyl pyrophosphate (IPP) to give C20geranylgeranyl pyrophosphate (GGPP). Two molecules of GGPP are condensedto synthesize a colorless phytoene as an initial carotenoid. Thephytoene is converted into phytofluene, ζ-carotene, neurosporene andthen lycopene by a series of desaturation reactions, and lycopene is inturn converted into β-carotene by the cyclization reaction. It isbelieved that a variety of xanthophylls are synthesized by introducing ahydroxyl group or a keto group into the β-carotene (See Britton, G.,"Biosynthesis of Carotenoids"; Plant Pigments, Goodwin, T. W. ed.,London, Academic Press, 1988, pp. 133-182).

The present inventors have recently made it possible to clone acarotenoid biosynthesis gene cluster from a epiphytic non-photosyntheticbacterium Erwinia uredovora in Escherichia coli with an index of theyellow tone of the bacterium, a variety of combinations of the genesbeing expressed in microorganisms such as Escherichia coli to producephytoene, lycopene, β-carotene, and zeaxanthin which is a derivative ofβ-carotene into which hydroxyl groups have been introduced (See FIG. 10;Misawa, N., Nakagawa, M., Kobayashi, K., Yamano, S., Izawa, Y.,Nakamura, K., Harashima, K.; "Elucidation of the Erwinia uredovoraCarotenoid biosynthetic Pathway by Functional Analysis of Gene ProductsExpressed in Escherichia coli", J. Bacteriol., 172, p.6704-6712, 1990;Misawa, N., Yamano, S., Ikenaga, H., "Production of β-carotene inZymomonas mobilis and Agrobacterium tumefaciencs by Introduction of theBiosynthesis Genes from Erwinia uredovora", Appl. environ. Microbiol.,57, p. 1847-1849, 1991; and Japanese Patent Application No. 58786/1991(Japanese Patent Application No. 53255/1990): "DNA Strands useful forthe Synthesis of Carotenoids").

On the other hand, astaxanthin, a red xanthophyll, is a typical animalcarotenoid which occurs particularly in a wide variety of marine animalsincluding red fishes such as a sea bream and a salmon, and crustaceanssuch as a crab and a lobster. In general, animals cannot biosynthesizecarotenoids, so that it is necessary for them to ingest carotenoidssynthesized by microorganisms or plants from their environments. Thus,astaxanthin has hitherto been used widely for strengthening the color ofcultured fishes and shellfishes such as a sea bream, a salmon, a lobsterand the like. Moreover, astaxanthin has attracted attention not only asa coloring matter in foods but also as an anti-oxidant for removingactive oxygen generated in bodies, which causes carcinoma (see TakaoMatsuno ed., "Physiological Functions and Bioactivities of Carotenoidsin Animals", Kagaku to Seibutsu, 28, p. 219-227, 1990). As the sourcesof astaxanthin, there have been known crustaceans such as a krill in theAntarctic Ocean, cultured products of a yeast Phaffia, cultured productsof a green alga Haematococcus, and products obtained by the organicsynthetic methods. However, when crustaceans such as a krill in theAntarctic Ocean or the like are used, it requires laborious works andmuch expenses for the isolation of astaxantin from contaminants such aslipids and the like during the harvesting and extraction of the krill.Moreover, in the case of the cultured product of the yeast Phaffia, agreat deal of expenses are required for the gathering and extraction ofastaxanthin, since the yeast has rigid cell walls and producesastaxanthin only in a low yield. Also, in the case of the culturedproduct of the green alga Haematococcus, not only a location forcollecting sunlight or an investment of a culturing apparatus forsupplying an artificial light is required in order to supply light whichis essential to the synthesis of astaxantin, but also it is difficult toseparate astaxanthin from fatty acid esters as by-products orchlorophylls present in the cultured products. From these reasons,astaxanthin produced from biological sources is in the present situationinferior to that obtained by the organic synthetic methods on the basisof cost. The organic synthetic methods however have a problem ofby-products produced during the reactions in consideration of its use asa feed for fishes and shellfishes and an additive to foods, and theproducts obtained by the organic synthetic methods are opposed to theconsumer's preference for natural products. Thus, it has been desired tosupply an inexpensive astaxanthin which is safe and produced frombiological sources and thus has a good image to consumers and to developa process for producing the astaxanthin.

DISCLOSURE OF THE INVENTION

It would be considered very useful to find a group of genes for playinga role of the biosynthesis of astaxanthin, because it is possible toafford astaxanthin-producing ability to a microorganism optimum insafety as a food or in potentiality for producing astaxanthin,regardless of the presence of astaxanthin-producing ability, byintroducing a gene cluster for astaxanthin biosynthesis into themicroorganism. No problem of by-products as contaminants is caused inthis case, so that it would be considered not so difficult to increasethe production amount of astaxanthin with a recent advanced technique ofgene manipulation to a level higher than that accomplished by theorganic synthetic methods. However, the groups of genes for synthesizingzeaxanthin, one of the xanthophylls, have already been acquired by thepresent inventors as described above, while no genes encoding a ketogroup-introducing enzyme required for the synthesis of astaxanthin havenot successfully obtained. The reason of the failure in obtaining thegenes includes that the keto group-introducing enzyme is a membraneprotein and loses its activity when isolated from the membrane, so thatit was impossible to purify the enzyme or measure its activity and noinformation on the enzyme has been obtained. Thus, it has hitherto beenimpossible to produce astaxanthin in microorganisms by genemanipulation.

The object of the present invention is to provide DNA strands whichcontain genes required for producing keto group-containing xanthophylls(ketocarotenoids) such as astaxanthin in microorganisms by obtainingsuch genes coding for enzymes such as a keto group-introducing enzymerequired for producing keto group-containing xanthophylls(ketocarotenoids) such as astaxanthin, and to provide a process forproducing keto group-containing xanthophylls (ketocarotenoids) such asastaxanthin with the microorganisms into which the DNA strands have beenintroduced.

The gene cloning method which is often used usually comprising purifyingthe aimed protein, partially determining the amino acid sequence andobtaining genes by a synthetic probe cannot be employed because of thepurification of the astaxanthin synthetic enzyme being impossible asdescribed above. Thus, the present inventors have paid attention to thefact that the cluster of carotenoid synthesis genes innon-photosynthetic bacterium (Erwinia) functions in Escherichia coli, inwhich lycopene and β-carotene which are believed to be intermediates forbiosynthesis of astaxanthin are allowed to produce with combinations ofthe genes from the gene cluster, and have used Escherichia coli as ahost for cloning of astaxanthin synthetic genes. The present inventorshave also paid attention to the facts that some marine bacteria have anastaxanthin-producing ability (Yokoyama, A., Izumida, H., Miki, W.,"Marine bacteria produced astaxanthin", 10th International Symposium onCarotenoids, Abstract, CL11-3, 1993), that a series of related geneswould constitute a cluster in the case of bacteria, and that the genecluster would be expressed functionally in Escherichia coli in the caseof bacteria. The present inventors have thus selected the marinebacteria as the gene sources. They have carried out researches with acombination of these two means and successfully obtained the gene groupwhich is required for the biosynthesis of astaxanthin and the other ketogroup-containing xanthophylls from marine bacteria. They have thusaccomplished the present invention. In addition, it has been firstelucidated in the present invention that the astaxanthin synthesis genecluster in marine bacteria constitutes a cluster and expresses itsfunction in Escherichia coli, and these gene products can utilizeβ-carotene or lycopene as a substrate.

The DNA strands according to the present invention are set forth asfollows.

(1) A DNA strand having a nucleotide sequence which encodes apolypeptide having an enzyme activity for converting the methylene groupat the 4-position of the β-ionone ring into a keto group.

(2) A DNA strand having a nucleotide sequence which encodes apolypeptide having an enzyme activity for converting the methylene groupat the 4-position of the β-ionone ring into a keto group and having anamino acid sequence substantially of amino acid Nos. 1-212 which isshown in the (SEQ ID NO: 2).

(3) A DNA strand hybridizing the DNA strand described in (2) and havinga nucleotide sequence which encodes a polypeptide having an enzymeactivity described in (2).

(4) A DNA strand having a nucleotide sequence which encodes apolypeptide having an enzyme activity for converting the methylene groupat the 4-position of the β-ionone ring into a keto group and having anamino acid sequence substantially of amino acid Nos. 1-242 which isshown in the (SEQ ID NO: 9).

(5) A DNA strand hybridizing the DNA strand described in (4) and havinga nucleotide sequence which encodes a polypeptide having an enzymeactivity described in (4).

(6) A DNA strand having a nucleotide sequence which encodes apolypeptide having an enzyme activity for converting β-carotene intocanthaxanthin via echinenone and having an amino acid sequencesubstantially of amino acid Nos. 1-212 which is shown in the (SEQ ID NO:5).

(7) A DNA strand hybridizing the DNA strand described in (6) and havinga nucleotide sequence which encodes a polypeptide having an enzymeactivity described in (6).

(8) A DNA strand having a nucleotide sequence which encodes apolypeptide having an enzyme activity for converting β-carotene intocanthaxanthin via echinenone and having an amino acid sequencesubstantially of amino acid Nos. 1-242 which is shown in the (SEQ ID NO:9).

(9) A DNA strand hybridizing the DNA strand described in (8) and havinga nucleotide sequence which encodes a polypeptide having an enzymeactivity described in (8).

(10) A DNA strand having a nucleotide sequence which encodes apolypeptide having an enzyme activity for converting the methylene groupat the 4-position of the 3-hydroxy-β-ionone ring into a keto group.

(11) A DNA strand having a nucleotide sequence which encodes apolypeptide having an enzyme activity for converting the methylene groupat the 4-position of the 3-hydroxy-β-ionone ring into a keto group andhaving an amino acid sequence substantially of amino acid Nos. 1-212which is shown in the (SEQ ID NO: 2).

(12) A DNA strand hybridizing the DNA strand described in (11) andhaving a nucleotide sequence which encodes a polypeptide having anenzyme activity described in (11).

(13) A DNA strand having a nucleotide sequence which encodes apolypeptide having an enzyme activity for converting the methylene groupat the 4-position of the 3-hydroxy-β-ionone ring into a keto group andhaving an amino acid sequence substantially of amino acid Nos. 1-242which is shown in the (SEQ ID NO: 9).

(14) A DNA strand hybridizing the DNA strand described in (13) andhaving a nucleotide sequence which encodes a polypeptide having anenzyme activity described in (13).

(15) A DNA strand having a nucleotide sequence which encodes apolypeptide having an enzyme activity for converting zeaxanthin intoastaxanthin by way of 4-ketozeaxanthin and having an amino acid sequencesubstantially of amino acid Nos. 1-212 which is shown in the (SEQ ID NO:2).

(16) A DNA strand hybridizing the DNA strand described in (15) andhaving a nucleotide sequence which encodes a polypeptide having anenzyme activity described in (15).

(17) A DNA strand having a nucleotide sequence which encodes apolypeptide having an enzyme activity for converting zeaxanthin intoastaxanthin by way of 4-ketozeaxanthin and having an amino acid sequencesubstantially of amino acid Nos. 1-242 which is shown in the (SEQ ID NO:9).

(18) A DNA strand hybridizing the DNA strand described in (17) andhaving a nucleotide sequence which encodes a polypeptide having anenzyme activity described in (17).

(19) A DNA strand having a nucleotide sequence which encodes apolypeptide having an enzyme activity for adding a hydroxyl group to the3-carbon of the 4-keto-β-ionone ring.

(20) A DNA strand having a nucleotide sequence which encodes apolypeptide having an enzyme activity for adding a hydroxyl group toposition 3-carbon of the 4-keto-β-ionone ring and having an amino acidsequence substantially of amino acid Nos. 1-162 which is shown in the(SEQ ID NO: 4).

(21) A DNA strand hybridizing the DNA strand described in (20) andhaving a nucleotide sequence which encodes a polypeptide having anenzyme activity described in (20).

(22) A DNA strand having a nucleotide sequence which encodes apolypeptide having an enzyme activity for adding a hydroxyl group toposition 3-carbon of the 4-keto-β-ionone ring and having an amino acidsequence substantially of amino acid Nos. 1-162 which is shown in the(SEQ ID NO: 11).

(23) A DNA strand hybridizing the DNA strand described in (22) andhaving a nucleotide sequence which encodes a polypeptide having anenzyme activity described in (22).

(24) A DNA strand having a nucleotide sequence which encodes apolypeptide having an enzyme activity for converting canthaxanthin intoastaxanthin by way of phoenicoxanthin and having an amino acid sequencesubstantially of amino acid Nos. 1-162 which is shown in the (SEQ ID NO:4).

(25) A DNA strand hybridizing the DNA strand described in (24) andhaving a nucleotide sequence which encodes a polypeptide having anenzyme activity described in (24).

(26) A DNA strand having a nucleotide sequence which encodes apolypeptide having an enzyme activity for converting canthaxanthin intoastaxanthin by way of phoenicoxanthin and having an amino acid sequencesubstantially of amino acid Nos. 1-162 which is shown in the (SEQ ID NO:11).

(27) A DNA strand hybridizing the DNA strand described in (26) andhaving a nucleotide sequence which encodes a polypeptide having anenzyme activity described in (26).

The present invention also relates to a process for producingxanthophylls.

That is, the process for producing xanthophylls according to the presentinvention is set forth below.

(1) A process for producing a xanthophyll comprising introducing the DNAstrand described in any one of the above mentioned DNA strands (1)-(9)into a microorganism having a β-carotene-synthesizing ability, culturingthe transformed microorganism in a culture medium, and obtainingcanthaxanthin or echinenone from the cultured cells.

(2) A process for producing a xanthophyll comprising introducing the DNAstrand described in any one of the above mentioned DNA strands (10)-(18)into a microorganism having a zeaxanthin-synthesizing ability, culturingthe transformed microorganism in a culture medium, and obtainingastaxanthin or 4-ketozeaxanthin from the cultured cells.

(3) A process for producing a xanthophyll comprising introducing the DNAstrand described in any one of the above mentioned DNA strands (19)-(27)into a microorganism having a canthaxanthin-synthesizing ability,culturing the transformed microorganism in a culture medium, andobtaining astaxanthin or phoenicoxanthin from the cultured cells.

(4) A process for producing a xanthophyll according to any one of theabove mentioned processes (1)-(3), wherein the microorganism is abacterium or yeast.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates diagrammatically the nucleotide sequence of the ketogroup-introducing enzyme gene (crt W gene) of the marine bacteriumAgrobacterium aurantiacus sp. nov. MK1 and the amino acid sequence of apolypeptide to be encoded thereby (SEQ ID NOS: 1-2).

FIG. 2 illustrates diagrammatically the nucleotide sequence of thehydroxyl group-introducing enzyme gene (crt Z gene) of the marinebacterium Agrobacterium aurantiacus sp. nov. MK1 and the amino acidsequence of a polypeptide to be encoded thereby (SEQ ID NOS: 3-4).

FIG. 3 illustrates diagrammatically the nucleotide sequence of thelycopene-cyclizing enzyme gene (crt Y gene) of the marine bacteriumAgrobacterium aurantiacus sp. nov. MK1 and the amino acid sequence of apolypeptide to be encoded thereby (SEQ ID NOS: 5-6).

FIG. 4 illustrates diagrammatically the continuation of the sequencesfollowing to those illustrated in FIG. 3 (SEQ ID NOS: 5-6).

FIG. 5 illustrates diagrammatically the nucleotide sequence of thexanthophyll synthesis gene cluster of the marine bacterium Agrobacteriumaurantiacus sp. nov. MK1.

The letters A-F in FIG. 5 correspond to those in FIGS. 1-4.

FIG. 6 illustrates diagramatically the continuation of the sequencefollowing to that illustrated in FIG. 5 (SEQ ID NO: 7).

FIG. 7 illustrates diagrammatically the continuation of the sequencefollowing to that illustrated in FIG. 6 (SEQ ID NO: 7).

FIG. 8 illustrates diagrammatically the continuation of the sequencefollowing to that illustrated in FIG. 7 (SEQ ID NO: 7).

FIG. 9 illustrates diagrammatically the continuation of the sequencefollowing to that illustrated in FIG. 8 (SEQ ID NO: 7).

FIG. 10 illustrates diagrammatically the carotenoid biosynthetic routeof the non-photosynthesis bacterium Erwinia uredovora and the functionsof the carotenoid synthetic genes.

FIG. 11 illustrates diagrammatically the main xanthophyll biosyntheticroutes of marine bacteria Agrobacterium aurantiacus sp. nov. MK1 andAlcaligenes sp. PC-1 and the functions of the xanthophyll synthesisgenes.

The function of crtY gene, however, has been confirmed only in theformer bacterium.

FIG. 12 illustrates diagrammatically a variety of deletion plasmidscontaining the xanthophyll synthesis genes (cluster) of the marinebacterium Agrobacterium aurantiacus sp. nov. MK1.

The letter represents the promoter of the lac of the vector pBluescriptII SK. The positions of cutting with restriction enzymes are representedby abbreviations as follows: Sa, SacI; X, XbaI;B, BamHI; P, PstI; E,EcoRI; S, SalI; A, ApaI; K, KpnI; St, StuI; N, NruI; Bg, BglII; Nc,NcoI; Hc, HincII.

FIG. 13 illustrates diagrammatically the nucleotide sequence of the ketogroup-introducing enzyme gene (crtW gene) of the marine bacteriumAlcaligenes sp. PC-1 and the amino acid sequence of a polypeptide to beencoded thereby (SEQ ID NOS: 8-9).

FIG. 14 illustrates diagrammatically the continuation of the sequencesfollowing to those illustrated in FIG. 13 (SEQ ID NOS: 8-9).

FIG. 15 illustrates diagrammatically the nucleotide sequence of thehydroxyl group-introducing enzyme gene (crtZ gene) of the marinebacterium Alcaligenes sp. PC-1 and the amino acid sequence of apolypeptide to be encoded thereby (SEQ ID NOS: 10-11).

FIG. 16 illustrates diagrammatically the nucleotide sequence of thexanthophyll synthetic gene cluster of the marine bacterium Alcaligenessp. PC-1 and the amino acid sequence of a polypeptide to be encodedthereby (SEQ ID NO: 12). The letters A-D in FIG. 16 correspond to thosein FIGS. 13-15.

FIG. 17 illustrates diagrammatically the continuation of the sequencesfollowing to those illustrated in FIG. 16 (SEQ ID NO: 12).

FIG. 18 illustrates diagrammatically the continuation of the sequencesfollowing to those illustrated in FIG. 17 (SEQ ID NO: 12).

FIG. 19 illustrates diagrammatically a variety of deletion plasmidscontaining the xanthophyll synthetic genes (cluster) of the marinebacterium Alcaligenes sp. PC-1.

The letter represents the promoter of the lac of the vector pBluescriptII SK+.

FIG. 20 illustrates diagrammatically xanthophyll biosynthetic routescontaining miner biosynthetic routes in the marine bacteriaAgrobacterium aurantiacus sp. no. MK1 and Alcaligenes sp. PC-1 and thefunctions of the xanthophyll synthesis genes.

Miner biosynthetic routes are represented by dotted arrows.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention is intended to provide DNA strands which areuseful for synthesizing a keto group-containing xanthophylls(ketocarotenoids) such as astaxanthin derived from a marine bacteriaAgrobacterium aurantiacus sp. nov. MK1 and Alcaligenes sp. PC-1, and aprocess for producing keto group-containing xanthophylls(ketocarotenoids), i.e. astaxanthin, phoenicoxanthin, 4-ketozeaxanthin,canthaxanthin, and echinenone with use of a microorganism into which theDNA strands have been introduced.

The DNA strands according to the present invention are in principleillustrated generally by the aforementioned DNA strands (1), (10) and(19) from the standpoint of the fine chemical-generating reaction, andbasically defined by the aforementioned DNA strands (2), (4), (11),(13), (20) and (22). The specific examples of the DNA strands (2) and(4) are the aforementioned DNA strands (6) and (8); the specificexamples of the DNA strands (11) and (13) are the aforementioned DNAstrands (15) and (17); and the specific examples of the DNA strands (20)and (22) are the aforementioned DNA strands (24) and (26). In thisconnection, the DNA strands (3), (5), (7), (9), (12), (14), (16), (18),(21), (23), (25) and (27) hybridize the DNA strands (2), (4), (6), (8),(11), (13), (15), (17), (20), (22), (24) and (26), respectively, under astringent condition.

The polypeptides encoded by the DNA strands according to the presentinvention have amino acid sequences substantially in a specific range asdescribed above in SEQ ID NOS: 2, 4, 9 and 11 (FIGS. 1-2, and 13-15),e.g. an amino acid sequence of amino acid Nos. 1-212 in SEQ ID NOS: 2(A-B in FIG. 1). In the present invention, four polypeptides encoded bythese DNA strands, that is four enzymes participating in thexanthophyll-producing reaction) may be modified by deletion,substitution or addition in some of the amino acids provided that thepolypeptides have the enzyme activities as described above (see Example13). This corresponds to that "amino acid sequences . . . substantially. . . " For instance, an enzyme of which amino acid at the firstposition (Met) has been deleted is also involved in the polypeptide orenzyme obtained by the modification of the amino acid sequence. In thisconnection, it is needless to say that the DNA strands according to thepresent invention for encoding the polypeptides also include, inaddition to those having nucleotide sequences in a specific range shownin SEQ ID NOS: 2, 4, 9 and 11 (FIGS. 1-2, and 13-15), degenerate isomersencoding the same polypeptides as above except degenerate codons.

Keto Group-introducing Enzyme Gene (crtW)

The DNA strands (1)-(18) are genes which encode the ketogroup-introducing enzymes (referred to hereinafter as crtW). Typicalexamples of the genes are crtW genes cloned from the marine bacteriaAgrobacterium aurantiacus sp. nov. MK1 or Alcaligenes sp. PC-1, whichare the DNA strands comprising the nucleotide sequences encoding thepolypeptides having the amino acid sequences A-B in FIG. 1 (amino acidNo: 212 in SEQ ID NO: 1) or A-B in FIGS. 13-14 (amino acid Nos. 1-242 inSEQ ID NO: 9). The crtW gene product (also referred to hereinafter asCrtW) has an enzyme activity for converting the 4-methylene group of theβ-ionone ring into a keto group, and one of the specific examples is anenzyme activity for synthesizing canthaxanthin with β-carotene as asubstrate by way of echinenone (see FIG. 11). In addition, the crtW geneproduct also has an enzyme activity for converting the 4-methylene groupof the 3-hydroxy-β-ionone ring into a keto group, and one of thespecific examples is an enzyme activity for synthesizing astaxanthinwith zeaxanthin as a substrate by way of 4-ketozeaxanthin (see FIG. 11).In this connection, the polypeptides having such enzyme activities andthe DNA strands encoding the polypeptides have not hitherto beenreported, and the polypeptides or the DNA strands encoding thepolypeptides has no overall homology to polypeptides or DNA strandswhich have hitherto been reported. Moreover, no such information hasbeen reported that one enzyme has an activity to convert directly amethylene group of not only the β-ionone ring and the 3-hydroxy-β-iononering but also the other compounds into a keto group. Moreover, ahomology of CrtW as high as 83% identity at an amino acid sequence levelwas shown between Agrobacterium and Alcaligenes.

On the other hand, it is possible to allow a microorganisms such asEscherichia coli or the like to produce β-carotene or zeaxanthin byusing the carotenoid synthesis genes of the non-photosynthetic bacteriumErwinia, that is the crtE, crtB, crtI and crtY genes of Erwinia affordthe microorganism such as Escherichia coli or the like theβ-carotene-producing ability, and the crtE, crtB, crtI, crtY and crtZgenes of Erwinia afford the microorganisms such as Escherichia coli orthe like the zeaxanthin-producing ability (see FIG. 10 and Laid-OpenPublication of WO91/13078). Thus, the substrate of CrtW is supplied bythe crt gene cluster of Erwinia, so that when additional crtW gene isintroduced into the microorganism such as Escherichia coli or the likewhich contains the aforementioned crt gene cluster of Erwinia, theβ-carotene-producing microorganism will produce canthaxanthin by way ofechinenone, and the zeaxanthin-producing microorganism will produceastaxanthin by way of 4-ketozeaxanthin.

Hydroxyl Group-introducing Enzyme Gene (crtZ)

The DNA strands (19)-(27) are genes encoding a hydroxylgroup-introducing enzyme (referred to hereinafter as crtZ). Typicalexamples of the genes are crtZ genes cloned from the marine bacteriaAgrobacterium aurantiacus sp. nov. MK1 or Alcaligenes sp. PC-1, whichare the DNA strands comprising the nucleotide sequences encoding thepolypeptides having the amino acid sequences C-D in FIG. 2 (amino acidNos. 1-162 in SEQ ID NO: 4) or C-D in FIGS. 15 (amino acid Nos. 1-162 inSEQ ID NO: 11). The crtZ gene product (also referred to hereinafter asCrtZ) has an enzyme activity for adding a hydroxyl group to the 3-carbonatom of the β-ionone ring, and one of the specific examples is an enzymeactivity for synthesizing zeaxanthin with use of β-carotene as asubstrate by way of β-cryptoxanthin (see FIG. 11). In addition, the crtZgene product also has an enzyme activity for adding a hydroxyl group tothe 3-carbon atom of the 4-keto-β-ionone ring, and one of the specificexamples is an enzyme activity for synthesizing astaxanthin withcanthaxanthin as a substrate by way of phoenicoxanthin (see FIG. 11). Inthis connection, the polypeptide having the latter enzyme activity andthe DNA strand encoding the polypeptide have not hitherto been reported.Moreover, CrtZ of Agrobacterium and Alcaligenes showed a high homologywith CrtZ of Erwinia uredovora (57% and 58% identity), respectively, atan amino acid sequence level. Also, a high homology of 90% identity atan amino acid sequence level was shown between the CrtZ of Agrobacteriumand Alcaligenes.

It has been described above that it is possible to allow a microorganismsuch as Escherichia coli or the like to produce β-carotene by using thecarotenoid synthetic genes of the non-photosynthetic bacterium Erwinia.Moreover, it has been described above that it is possible to allow amicroorganism such as Escherichia coli or the like to producecanthaxanthin by adding crtW thereto. Thus, the substrate of CrtZ ofAgrobacterium or Alcaligenes is supplied by the crtE, crtB, crtI andcrtY genes of Erwinia (production of β-carotene), and the crtW gene ofAgrobacterium or Alcaligenes added thereto, so that when the crtZ geneof Agrobacterium or Alcaligenes is introduced into a microorganism suchas Escherichia coli or the like containing the crt gene group, theβ-carotene-producing microorganism will produce zeaxanthin by way ofβ-cryptoxanthin, and the canthaxanthin-producing microorganism willproduce astaxanthin by way of phoenicoxanthin.

Lycopene-cyclizing Enzyme Gene (crtY)

The DNA strand encoding the amino acid sequence substantially from E toF of FIGS. 3 and 4 (amino acid Nos. 1-386 in SEQ ID NO: 6) is a geneencoding a lycopene-cyclizing enzyme (referred to hereinafter as crtY).A typical example of the gene is the crtY gene cloned from the marinebacterium Agrobacterium aurantiacus sp. nov. MK1, which is the DNAstrand comprising the nucleotide sequence encoding the polypeptidehaving the amino acid sequence E-F in FIGS. 3 and 4. The crtY geneproduct (also referred to hereinafter as CrtY) has an enzyme activityfor synthesizing β-carotene with lycopene as a substrate (see FIG. 11).It is possible to allow a microorganism such as Escherichia coli or thelike to produce lycopene by using a carotenoid biosynthesis genes of anon-photosynthetic bacterium Erwinia, that is the crtE, crtB and crtIgenes of Erwinia give a microorganism such as Escherichia coli or thelike a lycopene biosynthesis ability (see FIG. 10, and Laid-OpenPublication of WO91/13078). Thus, the substrate of the CrtY ofAgrobacterium is supplied by the crt gene group of Erwinia, so that whenthe crtY of Agrobacterium is introduced into a microorganism such asEscherichia coli or the like containing the crt gene group, it ispossible to allow the microorganism to produce β-carotene.

In this connection, the CrtY of Agrobacterium has a significant homologyof 44.3% identity to the CrtY of Erwinia uredovora at the amino acidsequence level, and these CrtY enzymes also have the same enzymaticfunction (see FIGS. 10 and 11).

Bacteriological Properties of Marine Bacteria

The marine bacteria Agrobacterium aurantiacus sp. nov. MK1 andAlcaligenes sp. PC-1 as the sources of the xanthophyll synthetic genesshow the following bacteriological properties.

<Agrobacterium aurantiacus sp. nov. MK1>

(1) Morphology `Form and size of bacterium: rod, 0.9 μm×1.2 μm;

Motility: yes;

Flagellum: peripheric flagellum;

Polymorphism of cell: none;

Sporogenesis: none;

Gram staining: negative.

(2) Growths in culture media

Broth agar plate culture: non-diffusive circular orange colonies havinga gloss are formed.

Broth agar slant culture: a non-diffusive orange band having a gloss isformed.

Broth liquid culture: homogeneous growth all over the culture mediumwith a color in orange.

Broth gelatin stab culture: growth over the surface around the stabpore.

(3) Physiological properties

Reduction of nitrate: positive;

Denitrification reaction: negative;

Formation of indole: negative;

Utilization of citric acid: negative;

Formation of pigments: fat-soluble reddish orange pigment;

Urease activity: negative;

Oxidase activity: positive;

Catalase activity: positive;

β-Glucosidase activity (esculin degradability): positive;

β-Galactosidase activity: positive;

Growth range: pH, 5-9; temperature, 10°-40° C.:

Behavior towards oxygen: aerobic;

Durability to seawater: positive;

O-F test: oxidation;

Anabolic ability of saccharides:

Positive: D-glucose, D-mannose, D-galactose, D-fructose, lactose,maltose, sucrose, glycogen, N-acetyl-D-glucosamine;

Negative: L-arabinose, D-mannitol, inositol, L-rhamnose, D-sorbitol;

Anabolic ability of organic acids:

Positive: lactate;

Negative: citrate, malate, gluconate, caprinate, succinate, adipate;

Anabolic ability of the other organic materials:

Positive: inosine, uridine, glucose-1-phosphate, glucose-6-phosphate;

Negative: gelatin, L-arginine, DNA, casein.

<Alcaligenes sp. PC-1>

(1) Morphology

Form and size of bacterium: short rod, 1.4 μm;

Motility: yes;

Flagellum: peripheric flagellum;

Polymorphism of cell: none;

Sporogenesis: none;

Gram staining: negative.

(2) Growths in culture media

Broth agar plate culture: non-diffusive circular orange colonies havinga gloss are formed.

Broth agar slant culture: a non-diffusive orange band having a gloss isformed.

Broth liquid culture: homogeneous growth all over the culture mediumwith a color in orange.

Broth gelatin stab culture: growth over the surface around the stabpore.

(3) Physiological properties

Formation of pigments: fat-soluble reddish orange pigment;

Oxidase activity: positive;

Catalase activity: positive;

Growth range: pH, 5-9; temperature, 10°-40° C.;

Behavior towards oxygen: aerobic;

Durability to seawater: positive;

O-F test: oxidation;

Degradability of gelatin: negative.

Xanthophyll Synthetic Gene Cluster of the Other Marine Bacteria

It has hitherto been reported that 16 marine bacteria have an ability tosynthesize ketocarotenoids such as astaxanthin and the like (Yokoyama,A., Izumida, H., Miki, W., "Marine bacteria produced astaxanthin", 10thInternational Symposium on Carotenoids, Abstract, CL11-3, 1993). Ifeither of the crt genes of the aforementioned marine bacteriaAgrobacterium aurantiacus sp. nov. MK-1 or Alcaligenes sp. PC-1 is usedas a probe, the gene cluster playing a role of the biosynthesis ofketocarotenoids such as astaxanthin and the like should be obtained fromthe other astaxanthin producing marine bacteria by using the homology ofthe genes. In fact, the present inventors have successfully obtained thecrtW and crtZ genes as the strongly hybridizing DNA fragments from thechromosomal DNA of Alcaligenes PC-1 with use of a DNA fragmentcontaining crtW and crtZ of Ag. aurantiacus sp. nov. MK1 as a probe (seeExamples as for the details). Furthermore, when Alteromonas SD-402 wasselected from the remaining 14 marine bacteria having an astaxanthinsynthetic ability and a chromosomal DNA was prepared therewith andsubjected to the Southern hybridization experiment with a DNA fragmentcontaining crtW and crtZ of Ag. aurantiacus sp. nov. MK1, the probehybridized with the bands derived from the chromosomal DNA of the marinebacteria. The DNA strands according to the present invention alsoincludes a DNA strand which hybridizes with the DNA strands (2), (4),(6), (8), (11), (13), (15), (17), (20), (22), (24) and (26).

Acquisition of DNA Strands

Although one of the methods for obtaining the DNA strand having anucleotide sequence which encodes the amino acid sequence of each enzymedescribed above is to chemically synthesize at least a part of thestrand length according to the method for synthesizing a nucleic acid,it is believed more preferable than the chemical synthetic method toobtain the DNA strand by using the total DNA having been digested withan appropriate restriction enzyme to prepare a library in Escherichiacoli, from which library the DNA strand is obtained by the methodsconventionally used in the art of genetic engineering such as ahybridization method with an appropriate probe (see the xanthophyllsynthetic gene cluster of the other marine bacteria).

Transformation of an Microorganism such as Escherichia coli and GeneExpression

A variety of xanthophylls can be prepared by introducing the present DNAstrands described above into appropriate microorganisms such asbacteria, for example Escherichia coli, Zymomonas mobilis andAgrobacterium tumefaciens, and yeasts, for example Saccharomycescerivisiae.

The outline for introducing an foreign gene into a preferredmicroorganism is described below.

The procedure or method for introducing and expressing the foreign genein a microorganism such as Escherichia coli or the like comprises theones usually used in the art of genetic engineering in addition to thosedescribed below in the present invention and may be carried outaccording to the procedure or method (see, e.g., "Vectors for CloningGenes", Methods in Enzymology, 216, p. 469-631, 1992, Academic Press,and "Other Bacterial Systems", Methods in Enzymology, 204, p. 305-636,1991, Academic Press).

<Escherichia coli>

The method for introducing foreign genes into Escherichia coli includesseveral efficient methods such as the Hanahan's method and the rubidiummethod, and the foreign genes may be introduced according to thesemethods (see, for example, Sambrook, J., Fritsch, E. F., Maniatis, T.,"Molecular Cloning--A Laboratory Manual", Cold Spring Harbor LaboratoryPress, 1989). While foreign genes in Escherichia coli may be expressedaccording to the conventional methods (see, for example, "MolecularCloning--A Laboratory Manual"), the expression can be carried out forexample with a vector for Escherichia coli having a lac promoter in thepUC or pBluescript series. The present inventors have used a vectorpBluescrip II SK or KS for Escherichia coli having a lac promoter andthe like to insert the crtW, crtZ and crtY genes of Agrobacteriumaurantiacus sp. nov. MK1 and the crtW and crtZ genes of Alcaligenes sp.PC-1 and allowed to express these genes in Escherichia coli.

<Yeast>

The method for introducing foreign genes into yeast Saccharomycescerivisiae includes the methods which have already been established suchas the lithium method and the like, and the introduction may be carriedout according to these methods (see, for example, Ed. Yuichi Akiyama,compiled by Bio-industry Association, "New Biotechnology of Yeast",published by IGAKU SHUPPAN CENTER). Foreign genes can be expressed inyeast by using a promoter and a terminator such as PGK and GPD toconstruct an expression cassette in which the foreign gene is insertedbetween the promoter and the terminator so that transcription is ledthrough, and inserting the expression cassette into a vector such as theYRp system which is a multi-copy vector for yeast having the ARSsequence of the yeast chromosome as the replication origin, the YEpsystem which is a multi-copy vector for yeast having the replicationorigin of the 2 μm DNA of yeast, and the YIp system which is a vectorfor integrating a yeast chromosome having no replication origin of yeast(see "New Biotechnology of Yeast", published by IGAKU SHUPPAN CENTER,ibid.; NIPPON NOGEI-KAGAKU KAI ABC Series "Genetic Engineering forProducing Materials", published by ASAKURA SHOTEN; and Yamano, S.,Ishii, T., Nakagawa, M., Ikenaga, H., Misawa, N., "Metabolic Engineeringfor Production of β-carotene and lycopene in Saccharomyces cerevisiae",Biosci. Biotech. Biochem., 58, p. 1112-1114, 1994).

<Zymomonas mobilis>

Foreign genes can be introduced into an ethanol-producing bacteriumZymomonas mobilis by the conjugal transfer method which is common toGram-negative bacteria, and the foreign genes can be expressed by usinga vector pZA22 for Zymomonas mobilis (see Katsumi Nakamura, "MolecularBreeding of Zymomonas mobilis", Nippon Nogei-Kagaku Kaishi, 63, p.1016-1018, 1989; and Misawa, N., Yamano, S., Ikanaga, H., "Production ofβ-Carotene in Zymomonas mobilis and Agrobacterium tumefaciens byIntroduction of the Biosynthesis Genes from Erwinia uredovora", Appl.Environ. Microbiol., 57, p.1847-1849, 1991).

<Agrobacterium tumefaciens>

Foreign genes can be introduced into a plant pathogenic bacteriumAgrobacterium tumefaciens by the conjugal transfer method which iscommon to Gram-negative bacteria, and the foreign genes can be expressedby using a vector pBI121 for a bacterium such as Agrobacteriumtumefaciens (see Misawa, N., Yamano, S., Ikenaga, H., "Production ofβ-Carotene in Zymomonas mobilis and Agrobacterium tumefaciens byIntroduction of the Biosynthesis Genes from Erwinia uredovora", Appl.Environ. Microbiol., 57, p. 1847-1849, 1991).

Production of Xanthophylls by Microorganisms

The gene cluster for the synthesis of ketocarotenoids such asastaxanthin derived from a marine bacterium can be introduced andexpressed by the procedure or method described above for introducing andexpressing an foreign gene in a microorganism.

Farnesyl pyrophosphate (FPP) is a substrate which is common not only tocarotenoids but also to other terpenoids such as sesquiterpenes,triterpenes, sterols, hopanols and the like. In general, microorganismssynthesize terpenoids even if they cannot synthesize carotenoids, sothat all of the microorganisms should basically have FPP as anintermediate metabolite. Furthermore, the carotenoid synthesis genecluster of a non-photosynthetic bacterium Erwinia has an ability tosynthesize the substrates of the crt gene products of Agrobacteriumaurantiacus sp. nov. MK1 or Alcaligenes sp. PC-1 by using FPP as asubstrate (see FIG. 10). The present inventors have already confirmedthat when the group of crt genes of Erwinia is introduced into not onlyEscherichia coli but also the aforementioned microorganisms, that is theyeast Saccharomyces cerevisiae, the ethanol producing bacteriumZymomonas mobilis, or the plant pathogenic bacterium Agrobacteriumtumefaciens, carotenoids such as β-carotene and the like can beproduced, as was expected, by these microorganisms (Yamano, S., Ishii,T., Nakagawa, M., Ikenaga, H., Misawa, N., "Metabolic Engineering forProduction of β-Carotene and Lycopene in Saccharomyces cerevisiae",Biosci. Biotech. Biochem., 58, p. 1112-1114, 1994; Misawa, N., Yamano,S., Ikenaga, H., "Production of β-Carotene in Zymomonas mobilis andAgrobacterium tumefaciens by Introduction of the Biosynthetic Genes fromErwinia uredovora", Appl. Environ. Microbiol., 57, p. 1847-1849, 1991;and Japanese Patent Application No. 58786/1991 (Japanese PatentApplication No. 53255/1990) by the present inventors: "DNA Strandsuseful for the Synthesis of Carotenoids").

Thus, it should be possible in principle to allow all of themicroorganisms, in which the gene introduction and expression system hasbeen established, to produce ketocarotenoids such as astaxanthin and thelike by introducing the combination of the carotenoid synthesis genecluster derived from Erwinia and the DNA strands according to thepresent invention (typically the carotenoid synthesis gene clusterderived from Agrobacterium aurantiacus sp. nov. MK1 or Alcaligenes sp.PC-1) at the same time into the same microorganism. The process forproducing a variety of ketocarotenoids in microorganisms are describedbelow.

<Production of canthaxanthin and echinenone>

It is possible to produce canthaxanthin as a final product andechinenone as an intermediate metabolite by introducing into amicroorganism such as Escherichia coli and expressing the crtE, crtB,crtI and crtY genes of Erwinia uredovora required for the synthesis ofβ-carotene and any one of the DNA strands of the present invention(1)-(9) which is a keto group-introducing enzyme gene (typically, thecrtW gene of Agrobacterium aurantiacus sp. nov. MK1 or AlcaligenesPC-1). The yields or the ratio of canthaxanthin and echinenone can bechanged by controlling the expression level of the DNA strand (crtWgene) or examining the culturing conditions of a microorganism havingthe DNA strand. Two embodiments in Escherichia coli are described below,and more details will be illustrated in Examples.

A plasmid pACCAR16ΔcrtX that a fragment containing the crtE, crtB, crtIand crtY genes of Erwinia uredovora has been inserted into theEscherichia coli vector pACYC184 and a plasmid pAK916 that a fragmentcontaining the crtW gene of Agrobacterium aurantiacus sp. nov. MK1 hasbeen inserted into the Escherichia coli vector pBluescript II SK- wereintroduced into Escherichia coli JM101 and cultured to the stationaryphase to collect bacterial cells and to extract carotenoid pigments. Theextracted pigments comprised 94% of canthaxanthin and 6% of echinenone.Also, canthaxanthin was obtained in a yield of 3 mg starting from 2liters of the culture solution.

A plasmid pACCAR16ΔcrtX that a fragment containing the crtE, crtB, crtIand crtY genes of Erwinia uredovora has been inserted into theEscherichia coli vector pACYC184 and a plasmid pPC17-3 that a fragmentcontaining the crtW gene of Alcaligenes PC-1 has been inserted into theEscherichia coli vector pBluescript II SK+ were introduced intoEscherichia coli JM101 and cultured to the stationary phase to collectbacterial cells and to extract carotenoid pigments. The extractedpigments comprised 40% of canthaxanthin and 50% of echinenone. Theremainder comprised 10% of unreacted β-carotene.

<Production of astaxanthin and 4-ketozeaxanthin>

It is possible to produce astaxanthin as a final product and4-ketozeaxanthin as an intermediate metabolite by introducing into amicroorganism such as Escherichia coli or the like and expressing thecrtE, crtB, crtI, crtY and crtZ genes of Erwinia uredovora required forthe synthesis of zeaxanthin and any one of the DNA strands of thepresent invention (10)-(18) which is a keto group-introducing enzymegene (typically, the crtW gene of Agrobacterium aurantiacus sp. nov. MK1or Alcaligenes PC-1). The yields or the ratio of astaxanthin and4-ketozeoxanthin can be changed by controlling the expression level ofthe DNA strand (crtW gene) or examining the culturing conditions of amicroorganism having the DNA strand.

Two embodiments in Escherichia coli are described below, and moredetails will be illustrated in Examples.

A plasmid pACCAR25ΔcrtX that a fragment containing the crtE, crtB, crtI,crtY and crtZ genes of Erwinia uredovora has been inserted into theEscherichia coli vector pACYC184 and a plasmid pAK916 that a fragmentcontaining the crtW gene of Ag. aurantiacus sp. nov. MK1 has beeninserted into the Escherichia coli vector pBluescript II SK- wereintroduced into Escherichia coli JM101 and cultured to the stationaryphase to collect bacterial cells and to extract carotenoid pigments. Theyield of the extracted pigments was 1.7 mg of astaxanthin and 1.5 mg of4-ketozeaxanthin based on 2 liters of the culture solution.

A plasmid pACCAR25ΔcrtX that a fragment containing the crtE, crtB, crtI,crtY and crtZ genes of Erwinia uredovora has been inserted into theEscherichia coli vector pACYC184 and a plasmid pPC17-3 that a fragmentcontaining the crtW gene of Alcaligenes PC-1 has been inserted into theEscherichia coli vector pBluescript II SK+ were introduced intoEscherichia coli JM101 and cultured to the stationary phase to collectbacterial cells and to extract carotenoid pigments. The yield of theextracted pigments was about 1 mg of astaxanthin and 4-ketozeaxanthin,respectively based on 2 liters of the culture solution.

<Production of astaxanthin and phoenicoxanthin>

It is possible to produce astaxanthin as a final product andphoenicoxanthin as an intermediate metabolite by introducing into amicroorganism such as Escherichia coli or the like and expressing thecrtE, crtB, crtI and crtY genes of Erwinia uredovora required for thesynthesis of β-carotene, any one of the DNA strands of the presentinvention (1)-(9) which is a keto group-introducing enzyme gene(typically, the crtW gene of Agrobacterium aurantiacus sp. nov. MK1 orAlcaligenes PC-1), and any one of the DNA strands of the presentinvention (19)-(27) which is a hydroxyl group-introducing enzyme gene(typically, the crtZ gene of Ag. aurantiacus sp. nov. MK1 or AlcaligenesPC-1). The yields or the ratio of astaxanthin and phoenicoxanthin can bechanged by controlling the expression level of the DNA strands (crtW andcrtZ genes) or examining the culturing conditions of a microorganismhaving the DNA strands. An embodiment in Escherichia coli are describedbelow, and more details will be illustrated in Examples.

A plasmid pACCAR16ΔcrtX that a fragment containing the crtE, crtB, crtIand crtY genes of Erwinia uredovora has been inserted into theEscherichia coli vector pACYC184 and a plasmid pAK96K that a fragmentcontaining the crtW and crtZ genes of Ag. aurantiacus sp. nov. MK1 hasbeen inserted into the Escherichia coli vector pBluescript II SK- wereintroduced into Escherichia coli JM101 and cultured to the stationaryphase to collect bacterial cells and to extract carotenoid pigments. Theyield of the extracted pigments comprised was 3 mg of astaxanthin and 2mg of phoenicoxanthin starting from 4 liters of the culture solution.

Deposition of Microorganisms

Microorganisms as the gene sources of the DNA strands of the presentinvention and Escherichia coli carrying the isolated genes (the DNAstrands of the present invention) have been deposited to NationalInstitute of Bioscience and Human Technology, Agency of IndustrialScience and Technology.

(i) Agrobacterium aurantiacus sp. nov. MK1 Deposition No: FERM BP-4506Entrusted Date: Dec. 20, 1993

(ii) Escherichia coli JM101 (pAccrt-EIB, pAK92) Deposition No: FERMBP-4505 Entrusted Date: Dec. 20, 1993

(iii) Alcaligense sp. PC-1 Deposition No: FERM BP-4760 Entrusted Date:Jul. 27, 1994

(iv) Escherichia coli β: pPC17 Deposition No: FERM BP-4761 EntrustedDate: Jul. 27, 1994

EXAMPLES

The present invention is further described more specifically withreference to the following examples without restriction of theinvention. In addition, the ordinary experiments of gene manipulationemployed herein is based on the standard methods (Sambrook, J., Fritsch,E. F., Maniatis, T., "Molecular Cloning--A Laboratory Manual", ColdSpring Harbor Laboratory Press, 1989), unless otherwise specified.

Example 1: Preparation of Chromosomal DNA

Chromosomal DNAs were prepared from three marine bacterial strains, i.e.Agrobacterium aurantiacus sp. nov. MK1, Alcaligenes sp. PC-1, andAlteromonas SD-402 (Yokoyama, A., Izumida, H., Miki, W., "Marinebacteria produced astaxanthin", 10th International Symposium onCarotenoids, Abstract, CL11-3, 1993). After each of these marinebacteria was grown in 200 ml of a culture medium (a culture mediumprepared according to the instruction of "Marine Broth" manufactured byDIFCO) at 25° C. for 4 days to the stationary phase, the bacterial cellswere collected, washed with a TES buffer (20 mM Tris, 10 mM EDTA, 0.1MNaCl, pH 8), subjected to heat treatment at 68° C. for 15 minutes, andsuspended into the solution I (50 mM glucose, 25 mM Tris, 10 mM EDTA, pH8) containing 5 mg/ml of lysozyme (manufactured by SEIKAGAKU KOGYO) and100 μg/ml of RNase A (manufactured by Sigma). After incubation of thesuspension at 37° C. for 1 hour, Proteinase K (manufactured byBoehringer-Mannheim) was added and the mixture was incubated at 37° C.for 10 minutes. After SARCOSIL (N-lauroylsarcosine Na, manufactured bySigma) was then added at the final concentration of 1% and the mixturewas sufficiently mixed, it was incubated at 37° C. for several hours.The mixture was extracted several times with phenol/chloroform, andethanol in a two-time amount was added slowly. Chromosomal DNA thusdeposited was wound around a glass rod, rinsed with 70% ethanol anddissolved in 2 ml of a TE buffer (10 mM Tris, 1 mM EDTA, pH 8) toprepare a chromosomal DNA solution.

Example 2: Preparation of Hosts for a Cosmid Library

(1) Preparation of phytoene-producing Escherichia coli

After the removal of the BstEII (1235)-Eco521 (4926) fragment from aplasmid pCAR16 having a carotenoid synthesis gene cluster except thecrtZ gene of Erwinia uredovora (Misawa, N.,Nakagawa, M., Kobayashi, K.,Yamano, S., Izawa, Y., Nakamura, K., Harashima, K., "Elucidation of theErwinia uredovora Carotenoid Biosynthetic Pathway by Functional Analysisof Gene Porducts expressed in Escherichia coli", J. Bacteriol., 172, p.6704-6712, 1990; and Japanese Patent Application No. 58786/1991(Japanese Patent Application No. 53255/1990): "DNA Strands useful forthe Synthesis of Carotenoids"), a 2.3 kb Asp718 (KpnI)-EcoRI fragmentcontaining the crtE and crtB genes required for the production ofphytoenes was cut out. This fragment was then inserted into the EcoRVsite of the E. coli vector pACYC184 to give an aimed plasmid(pACCRT-EB). The bacterium E. coli containing pACCRT-EB exhibitsresistance to an antibiotic chloramphenicol (Cm^(r)) and producesphytoenes (Linden, H., Misawa, N., Chamovitz, D., Pecker, I.,Hirschberg, J., Sandmann, G., "Functional Complementation in Escherichiacoli of Different Phytoene Desaturase Genes and Analysis of AccumulatedCarotenes", Z. Naturforsch., 46c, 1045-1051, 1991).

(2) Preparation of lycopene-producing Escherichia coli

After the removal of the BstEII (1235)-SnaBI (3497) fragment from aplasmid pCAR16 having a carotenoid synthesis gene cluster except thecrtZ gene of Erwinia uredovora, a 3.75 kb Asp718 (KpnI)-EcoRI fragmentcontaining the crtE, crtI and crtB genes required for the production oflycopene was cut out. This fragment was then inserted into the EcoRVsite of the E. coli vector pACYC184 to give an aimed plasmid(pACCRT-EIB). The bacterium E. coli containing pACCRT-EIB exhibitsCm^(r) and produces lycopene (Cunningham Jr, F. X., Chamovitz, D.,Misawa, N., Gatt, E., Hirschberg, J., "Cloning and Functional Expressionin Escherichia coli of Cyanobacterial Gene for Lycopene Cyclase, theEnzyme that catalyzes the Biosynthesis of β-Carotenes", FEBS Lett., 328,130-138, 1993).

(3) Preparation of β-carotene-producing Escherichia coli

After the crtX gene was inactivated by subjecting a plasmid pCAR16having a carotenoid synthesis gene cluster except the crtZ gene ofErwinia uredovora to digestion with restriction enzyme BstEII, theKlenow fragment treatment and the ligation reaction, a 6.0 kb Asp718(KpnI)-EcoRI fragment containing crtE, crtY, crtI and crtB genesrequired for the production of β-carotene was cut out. This fragment wasthen inserted into the EcoRV site of the E. coli vector pACYC184 to givean aimed plasmid (referred to hereinafter as pACCAR16ΔcrtX). Thebacterium E. coli containing pACCAR16ΔcrtX exhibits Cm^(r) and producesβ-carotene. In this connection, the restriction enzyme and enzymes usedfor genetic manipulation have been purchased from TAKARA SHUZO (K.K.) orBoehringer-Mannheim.

Example 3: Preparation of a Cosmid Library and Acquisition ofEscherichia coli which Exhibits Orange in Color

After the restriction enzyme Sau3AI was added in an amount of one unitto 25 μg of the chromosomal DNA of Agrobacterium aurantiacus sp. nov.MK1, the mixture was incubated at 37° C. for 15 minutes and heat treatedat 68° C. for 10 minutes to inactivate the restriction enzyme. Under thecondition, many partially digested fragments with Sau3AI were obtainedat about 40 kb. The cosmid vector pJBB (resistant to ampicillin(Ap^(r))) which had been subjected to BamHI digestion and alkalinephosphatase treatment and the right arm (shorter fragment) of pJBB whichhad been digested with SalI/BamHI and then recovered from the gel weremixed with a part of the above Sau3AI partial fragments, and ligated at12° C. overnight. In this connection, pJBB has been purchased fromAmersham.

Phage particles were obtained in an amount sufficient for preparing acosmid library by the in vitro packaging with a Gigapack Gold(manufactured by Stratagene; available from Funakoshi) using the DNAabove ligated.

After Escherichia coli DH1 (ATCC33849) and Escherichia coli DH1, each ofwhich has one of the three plasmids prepared in Example 2, were infectedwith the phage particles, these bacteria were diluted so that 100-300colonies were found on a plate, plated on LB containing appropriateantibiotics (1% trypton, 0.5% yeast extract, 1% NaCl), and cultured at37° C. or room temperature for a period of overnight to several days.

As a result, in cosmid libraries having the simple Escherichia coli(beige) or the phytoene-producing Escherichia coli (beige) withpACCRT-EB as a host, no colonies with changed color were obtainednotwithstanding the screening of a ten thousand or more of the coloniesfor respective libraries. On the other hand, in cosmid libraries havingthe lycopene-producing Escherichia coli (light red) with pACCRT-EIB orthe β-carotene-producing Escherichia coli (yellow) with pACCAR16ΔcrtX asa host, colonies exhibiting orange have appeared in a proportion of onestrain to several hundred colonies, respectively. Most of thesetransformed Escherichia coli strains which exhibits orange containedplasmid pJB8 in which about 40 kb partially digested Sau3AI -fragmentswere cloned. It is also understood from the fact that no colonies withchanged color appeared in cosmid libraries having the simple Escherichiacoli or the phytoene-producing Escherichia coli with pACCRT-EB as ahost, that Escherichia coli having an ability of producing a carotenoidsynthetic intermediate of the later steps of at least phytoene should beused as a host for the purpose of expression-cloning the xanthophyllsynthesis gene cluster from the chromosomal DNA of Agrobacteriumaurantiacus sp. nov. MK1.

Example 4: Localization of a Fragment Containing an Orange PigmentSynthesis Gene Cluster

When individual several ten colonies out of the orange colonies obtainedin cosmid libraries having the lycopene-producing Escherichia coli(light red) with pACCRT-EIB or the β-carotene-producing Escherichia coli(yellow) with pACCAR16ΔcrtX as a host were selected to analyze theplasmids, 33 kb-47 kb fragments partially digested with Sau3AI wereinserted in vector pJB8 in all of the colonies except one strain. Theremaining one strain (lycopene-producing Escherichia coli as a host)contains a plasmid, in which a 3.9 kb fragment partially digested withSau3AI was inserted in pJB8 (referred to hereinafter as plasmid pAK9).This was considered to be the one formed by the in vivo deletion of theinserted fragment after the infection to Escherichia coli. The samepigment (identified as astaxanthin in Example 6) as that in the orangecolonies obtained from the other cosmid libraries was successfullysynthesized with the lycopene-producing Escherichia coli having pAK9,pAK9 was used as a material in the following analyses.

Example 5: Determination of the Nucleotide Sequence in the OrangePigment Synthesis Gene Cluster

A 3.9 kb EcoRI inserted fragment prepared from pAK9 was inserted intothe EcoRI site of the Escherichia coli vector pBluescrip II SK+ to givetwo plasmids (pAK91 and pAK92) with the opposite directions of thefragment to the vector. The restriction enzyme map of one of theplasmids (pAK92) is illustrated in FIG. 12. When pAK92 was introducedinto the lycopene-producing Escherichia coli, orange colonies wereobtained as a result of the synthesis of astaxanthin (Example 6).However, no ability for synthesizing new pigments was afforded even ifpAK91 was introduced into the lycopene-producing Escherichia coli. Itwas thus considered that the pigment synthesis gene cluster in theplasmid pAK92 has the same direction as that of the lac promoter of thevector. Next, each of a 2.7 kb PstI fragment obtained by the PstIdigestion of pAK91, a 2.9 kb BamHI fragment obtained by the BamHIdigestion of pAK92, and 2.3 kb and 1.6 kb SalI fragments obtained by theSalI digestion of pAK92 was cloned into the vector pBluescrip II SK-.The restriction maps of plasmids referred to as pAK94, pAK96, pAK98,pAK910, pAK93, and pAK95 are illustrated in FIG. 12. The plasmids pAK94,pAK96, pAK98 and pAK910 have the pigment synthesis gene cluster in thesame direction as that of the lac promoter of the vector, while theplasmids pAK93 and pAK95 have the pigment synthesis gene cluster in theopposite direction to that of the promoter.

It was found that when the plasmid pAK96 having a 2.9 kb BamHI fragmentwas introduced into the lycopene-producing Escherichia coli, thetransformant also synthesized astaxanthin as in the case when theplasmid pAK92 having a 3.9 kb EcoRI fragment was introduced (Example 6),so that the DNA sequence of the 2.9 kb BamHI fragment was determined.

The DNA sequence was determined by preparing deletion mutants of the 2.9kb BamHI fragment from the normal and opposite directions anddetermining the sequence using clones having various lengths ofdeletions. The deletion mutants were prepared from the four plasmidspAK96, pAK98, pAK93 and pAK95 according to the following procedure: Eachof the plasmids, 10 μg, was decomposed with SacI and XbaI and extractedwith phenol/chloroform to recover DNA by ethanol precipitation. Each ofDNA was dissolved in 100 μl of ExoIII buffer (50 mM Tris-HCl, 100 mMNaCl, 5 mM MgCl₂, 10 mM 2-mercaptoethanol, pH 8.0), 180 units of ExoIIInuclease was added, and the mixture was maintained at 37° C. A 10 μlportion was sampled at every 1 minute, and two samples were transferredinto a tube in which 20 μl of MB buffer (40 mM sodium acetate, 100 mMNaCl, 2 mM ZnCl₂, 10% glycerol, pH 4.5) is contained and which is placedon ice. After completion of the sampling, five tubes thus obtained weremaintained at 65° C. for 10 minutes to inactivate the enzyme, five unitsof mung bean nuclease were added, and the mixtures were maintained at37° C. for 30 minutes. After the reaction, five DNA fragments differentfrom each other in the degrees of deletion were recovered for eachplasmid by agarose gel electrophoresis. The DNA fragments thus recoveredwas blunt ended with the Klenow fragment, subjected to the ligationreaction at 16° C. overnight, and Escherichia coli JM109 wastransformed. A single stranded DNA was prepared from each of variousclones thus obtained with a helper phage M13K07, and subjected to thesequence reaction with a fluorescent primer cycle-sequence kit availablefrom Applied Biosystem (K.K.), and the DNA sequence was determined withan automatic sequencer.

The DNA sequence comprising 2886 base pairs (bp) thus obtained isillustrated in FIGS. 5-9 (SEQ ID NO: 7). As a result of examining anopen reading frame having a ribosome binding site in front of theinitiation codon, three open reading frames which can encode thecorresponding proteins (A-B (nucleotide positions 229-864 of SEQ ID NO:7), C-D (nucleotide positions 864-1349), E-F (nucleotide positions1349-2506) in FIGS. 5-9) were found at the positions where the threexanthophyll synthesis genes crtW, crtZ and crtY are expected to bepresent. For the two open reading frames of A-B and E-F, the initiatingcodon is GTG, and for the remaining open reading frame C-D, it is ATG.

Example 6: Identification of the Orange Pigment

The lycopene-producing Escherichia coli JM101 having pAK92 or pAK96introduced thereinto (Escherichia coli (pACCRT-EIB, pAK92 or pAK96);exhibiting orange) or the β-carotene-producing Escherichia coli JM101having pAK94 or pAK96K (FIG. 12) introduced thereinto (Escherichia coli(pACCAR16ΔcrtX, pAK94 or pAK96K); exhibiting orange) was cultured in 4liters of a 2YT culture medium (1.6% trypton, 1% yeast extract, 0.5%NaCl) containing 150 μg/ml of ampicillin (Ap, manufactured by MeijiSeika) and 30 μg/ml of chloramphenicol (Cm, manufactured by Sankyo) at37° C. for 18 hours. Bacterial cells collected from the culture solutionwas extracted with 600 ml of acetone, concentrated, extracted twice with400 ml of chloroform/methanol (9/1), and concentrated to dryness. Then,thin layer chromatography (TLC) was conducted by dissolving the residuein a small amount of chloroform/methanol (9/1) and developing on asilica gel plate for preparative TLC manufactured by Merck withchloroform/methanol (15/1). The original orange pigment was separatedinto three spots at the Rf values of 0.72, 0.82 and 0.91 by TLC. Thepigment of the darkest spot at Rf 0.72 corresponding to 50% of the totalamount of orange pigment and the pigment of secondly darker spot at Rf0.82 were scratched off from the TLC plate, dissolved in a small amountof chloroform/methanol (9/1) or methanol, and chromatographed on aSephadex LH-20 column (15×300 mm) with an eluent of chloroform/methanol(9/1) or methanol to give purified materials in a yield of 3 mg (Rf0.72) and 2 mg (Rf 0.82), respectively.

It has been elucidated from the results of the UV-visible, ¹ H-NMR andFD-MS (m/e 596) spectra that the pigment at Rf 0.72 has the same planarstructure as that of astaxanthin. When the pigment was dissolved indiethyl ether:2-propanol:ethanol (5:5:2) to measure the CD spectrum, itwas proved to have stereochemical configuration of 3S, 3'S, and thusidentified as astaxanthin; see FIG. 11 for the structural formula).Also, the pigment at Rf 0.82 was identified as phoenicoxanthin (see FIG.11 for the structural formula) from the results of its UV-visible, ¹H-NMR and FD-MS (m/e 580) spectra. In addition, the pigment at 0.91 wascanthaxanthin (Example 7(2)).

Example 7: Identification of Metabolic Intermediates of Xanthophyll

(1) Identification of 4-ketozeaxanthin

The zeaxanthin producing Escherichia coli was prepared according to thefollowing procedure. That is to say, the plasmid pCAR25 having totalcarotenoid synthesis gene cluster of Er. uredorora (Misawa, N.,Nakagawa, M., Kobayashi, K., Yamano, S., Izawa, Y., Nakamura, K.,Harashima, K., "Elucidation of the Erwinia uredovora CarotenoidBiosynthetic Pathway by Functional Analysis of Gene Products expressedin Escherichia coli", J. Bacteriol., 172, p. 6704-6712, 1990; andJapanese Patent Application No. 58786/1991 (Japanese Patent ApplicationNo. 53255/1990): "DNA Strands useful for the Synthesis of Carotenoids")was digested with restriction enzyme BstEII, and subjected to the Klenowfragment treatment and ligation reation to inactivate the crtX gene byreading frame shift, and then a 6.5 kb Asp718 (KpnI)-EcoRI fragmentcontaining the crtE, crtY, crtI, crtB and crtZ genes required forproducing zeaxanthin was cut out. This fragment was then inserted intothe EcoRV site of the Escherichia coli vector pACYC184 to give the aimedplasmid (referred to hereinafter as pACCAR25ΔcrtX).

The zeaxanthin-producing Escherichia coli JM101 having pAK910 or pAK916(FIG. 12) introduced thereinto (Escherichia coli (pACCAR25ΔcrtX, pAK910or pAK916); exhibiting orange) was cultured in 2 liters of a 2YT culturemedium containing 150 μg/ml of Ap and 30 μg/ml of Cm at 37° C. for 18hours. Bacterial cells collected from the culture solution was extractedwith 300 ml of acetone, concentrated, extracted twice with 200 ml ofchloroform/methanol (9/1), and concentrated to dryness. Then, thin layerchromatography (TLC) was conducted by dissolving the residue in a smallamount of chloroform/methanol (9/1) and developing on a silica gel platefor preparative TLC manufactured by Merck with chloroform/methanol(15/1). The original orange pigment was separated into three spots atthe Rf values of 0.54 (46%), 0.72 (53%) and 0.91 (1%) by TLC. Thepigment at Rf 0.54 was scratched off from the TLC plate, dissolved in asmall amount of chloroform/methanol (9/1) or methanol, andchromatographed on a Sephadex LH-20 column (15×300 mm) with an eluent ofchloroform/methanol (9/1) or methanol to give a purified material in ayield of 1.5 mg.

This material was identified as 4-ketozeaxanthin (see FIG. 11 for thestructural formula) since its UV-visible spectrum, FD-MS spectrum (m/e582) and mobility in silica gel TLC (developed with chloroform/methanol(15/1)) accorded perfectly with those of the standard sample of4-ketozeaxanthin (purified from Agrobacterium aurantiacus sp. nov. MK1;Japanese Patent Application No. 70335/1993). In addition, the pigmentsat Rf 0.72 and 0.91 are astaxanthin (Example 6) and canthaxanthin(Example 7 (2)), respectively.

(2) Identification of canthaxanthin The β-carotene producing Escherichiacoli JM101 having pAK910 or pAK916 introduced thereinto (Escherichiacoli (pACCAR16ΔcrtX, pAK910 or pAK916); exhibiting orange) was culturedin 2 liters of a 2YT culture medium containing 150 μg/ml of Ap and 30μg/ml of Cm at 37° C. for 18 hours. Bacterial cells collected from theculture solution was extracted with 300 ml of acetone, concentrated,extracted twice with 200 ml of chloroform/methanol (9/1), andconcentrated to dryness. Then, thin layer chromatography (TLC) wasconducted by dissolving the residue in a small amount ofchloroform/methanol (9/1) and developing on a silica gel plate forpreparative TLC manufactured by Merck with chloroform/methanol (50/1).The pigment of the darkest spot corresponding to 94% of the total amountof orange pigments was scratched off from the TLC plate, dissolved in asmall amount of chloroform/methanol (9/1) or chloroform/methanol (1/1),and chromatographed on a Sephadex LH-20 column (15×300 mm) with aneluent of chloroform/methanol (9/1) or chloroform/methanol (1/1) to givea purified material in a yield of 3 mg.

This material was identified as canthaxanthin (see FIG. 11 for thestructural formula) since its UV-visible, ¹ H-NMR, FD-MS (m/e 564)spectra and mobility in silica gel TLC (Rf 0.53 on developing withchloroform/methanol (50/1)) accorded perfectly with those of thestandard sample of canthaxanthin (manufactured by BASF). In addition,the pigment corresponding to 6% of the total orange pigments found inthe initial extract was considered echinenone (see FIG. 11 for thestructural formula) on the basis of its UV-visible spectrum, mobility insilica gel TLC (Rf 0.78 on developing with chloroform/methanol (50/1)),and mobility in HPLC with NOVA PACK HR 6β C18 (3.9×300 mm; manufacturedby Waters) (RT 16 minutes on developing at a flow rate of 1.0 ml/minwith acetonitrile/methanol/2-propanol (90/6/4)).

(3) Identification of zeaxanthin

The β-carotene-producing Escherichia coli JM110 having pAK96NKintroduced thereinto (Escherichia coli (pACCAR16ΔcrtX, pAK96NK);exhibiting yellow) was cultured in 2 liters of a 2YT culture mediumcontaining 150 μg/ml of Ap and 30 μg/ml of Cm at 37° C. for 18 hours.Bacterial cells collected from the culture solution was extracted with300 ml of acetone, concentrated, extracted twice with 200 ml ofchloroform/methanol (9/1), and concentrated to dryness. Then, thin layerchromatography (TLC) was conducted by dissolving the residue in a smallamount of chloroform/methanol (9/1) and developing on a silica gel platefor preparative TLC manufactured by Merck with chloroform/methanol(9/1). The pigment of the darkest spot corresponding to 87% of the totalamount of yellow pigments was scratched off from the TLC plate,dissolved in a small amount of chloroform/methanol (9/1) or methanol,and chromatographed on a Sephadex LH-20 column (15×300 mm) with aneluent of chloroform/methanol (9/1) or methanol to give a purifiedmaterial in a yield of 3 mg.

It has been elucidated that this material has the same planar structureas that of zeaxanthin since its UV-visible, ¹ H-NMR, FD-MS (m/e 568)spectra and mobility in silica gel TLC (Rf 0.59 on developing withchloroform/methanol (9/1)) accorded perfectly with those of the standardsample of zeaxanthin (manufactured by BASF). When the pigment wasdissolved in diethyl ether:2-propanol:ethanol (5:5:2) to measure the CDspectrum, it was proved to have a stereochemical configuration of 3R,3'R, and thus identified as zeaxanthin (see FIG. 11 for the structuralformula). Also, the pigment corresponding to 13% of the total yellowpigments found in the initial extract was considered β-cryptoxanthin(see FIG. 11 for the structural formula) on the basis of its UV-visiblespectrum, mobility in silica gel TLC (Rf 0.80 on developing withchloroform/methanol (9/1)), and mobility in HPLC with NOVA PACK HR 6μC18 (3.9×300 mm; manufactured by Waters) (RT 19 minutes on developing ata flow rate of 1.0 ml/min with acetonitrile/methanol/2-propanol(90/6/4)).

(4) Identification of β-carotene

The lycopene-producing Escherichia coli JM101 having pAK98 introducedthereinto (Escherichia coli (pACCRT-EIB, pAK98); exhibiting yellow) wascultured in 2 liters of a 2YT culture medium containing 150 μg/ml of Apand 30 μg/ml of Cm at 37° C. for 18 hours. Bacterial cells collectedfrom the culture solution was extracted with 300 ml of acetone,concentrated, and extracted twice with 200 ml of hexane. The hexanelayer was concentrated and chromatographed on a silica gel column(15×300 mm) with an eluent of hexane/ethyl acetate (50/1) to give 3 mgof a purified material.

The material was identified as β-carotene (see FIG. 11 for thestructural formula), since all of the data of its UV-visible, FD-MSspectrum (m/e 536) and mobility in HPLC with NOVA PACK HR 6μ C18(3.9×300 mm; manufactured by Waters) (RT 62 minutes on developing at aflow rate of 1.0 ml/min with acetonitrile/methanol/2-propanol (90/6/4))accorded with those of the standard sample of β-carotene (all transtype; manufactured by Sigma).

Example 8: Identification of Xanthophyll Synthesis Gene Cluster

(1) Identification of a keto group-introducing enzyme gene

It is apparent from the results of Example 6 that among the 3.9 kbfragment contained in pAK9 (Example 4) or pAK92, all of the genesrequired for the synthesis of astaxanthin from lycopene is contained inthe 2.9 kb BamHI fragment at the right side (pAK96, FIG. 12). Thus, the1.0 kb fragment at the left side is not needed. Unique NcoI and KpnIsites are present within the 2.9 kb BamHI fragment of pAK96. It is foundfrom the results of Example 7 (3) that the 1.4 kb fragment (pAK96NK)between the NcoI and KpnI sites has a hydroxyl group-introducing enzymeactivity but has no keto group-introducing enzyme activity.Canthaxanthin can also be synthesized from β-carotene with the 2.9 kbBamHI fragment from which a fragment of the right side from unique SalIsite between the NcoI and KpnI sites had been removed (pAK910) or withthe 2.9 kb BamHI fragment from which a fragment of the right side fromthe HincII site positioned at the left side of the SalI site had beenremoved (pAK916), but activity for synthesizing canthaxanthin fromβ-carotene disappeared in the 2.9 kb BamHI fragment of pAK96 from whicha fragment of the right side from the NcoI site left of the HincII sitehad been removed. On the other hand, even if a fragment of the left sidefrom unique BglII site which is present leftward within the 0.9 kbBamHI-HincII fragment of pAK916 was removed, similar activity to that ofthe aforementioned BamHI-HincII fragment (pAK916) was observed. It isthus considered that a gene encoding a keto group-introducing enzymehaving an enzyme activity for synthesizing canthaxanthin from β-caroteneas a substrate is present within the 0.74 kb BglII-HincII fragment ofpAK916, and the aforementioned NcoI site is present within this gene. Asa result of determining the nucleotide sequence, an open reading framewhich corresponds to the gene and has a ribosome binding site just infront of the initiation codon was successfully detected, it was thendesignated as the crtW gene. The nucleotide sequence of the crtW geneand the encoded amino acid sequence are illustrated in FIG. 1 (SEQ IDNOS: 1-2).

The crtW gene product (CrtW) of Agrobacterium aurantiacus sp. nov. MK1has an enzyme activity for converting a methylene group at the4-position of a β-ionone ring into a keto group, and one of the specificexamples is an enzyme activity for synthesizing canthaxanthin fromβ-carotene as a substrate by way of echinenone (Example 7 (2); see FIG.11). Furthermore, the crtW gene product also has an enzyme activity forconverting a methylene group at the 4-position of a 3-hydroxy-β-iononering into a keto group, and one of the specific examples is an enzymeactivity for synthesizing astaxanthin from zeaxanthin as a substrate byway of 4-ketozeaxanthin (Example 7 (1); see FIG. 11). In addition,polypeptides having such enzyme activities and DNA strands encodingthese polypeptides have not hitherto been known, and the polypeptidesand the DNA strands encoding these polypeptides have no overall homologyto any polypeptides or DNA strands having been hitherto known. Also, nosuch informations have hitherto been described that a methylene group ofnot only a β-ionone ring and a 3-hydroxy-β-ionone ring but also theother compounds is directly converted into a keto group with an enzyme.

(2) Identification of a hydroxyl group-introducing enzyme gene

Unique SalI site is present within the 2.9 kb BamHI fragment of pAK96.When the 2.9 kb BamHI fragment is cut into two fragments at the SalIsite, these two fragments (pAK910 and pAK98) have no hydroxylgroup-introducing activity. That is to say, the left fragment (pAK910)has only a keto group-introducing enzyme activity (Example 7 (2)), andthe right fragment (pAK98) has only a lycopene-cyclizing enzyme activity(Example 7 (4)). On the other hand, when a 1.4 kb NcoI-KpnI fragment(pAK96NK) containing the aforementioned SalI site is introduced into aβ-carotene-producing Escherichia coli, zeaxanthin is synthesized by wayof β-cryptoxanthin (Example 7 (3)). It is thus considered that a geneencoding a hydroxyl group-introducing enzyme which has an enzymeactivity for synthesizing zeaxanthin from β-carotene as a substrate ispresent within the 1.4 kb NcoI-KpnI fragment of pAK96NK, and theaforementioned SalI site is present within this gene. As a result ofdetermining the nucleotide sequence, an open reading frame whichcorresponds to the gene and has a ribosome binding site just in front ofthe initiation codon was successfully detected, it was then referred toas the crtZ gene. The nucleotide sequence of the crtZ gene and theencoded amino acid sequence are illustrated in FIG. 2 (SEQ ID NOS: 3-4).

The crtZ gene product (CrtZ) of Agrobacterium aurantiacus sp. nov. MK1has an enzyme activity for adding a hydroxyl group to the 3-carbon of aβ-ionone ring, and one of the specific examples is an enzyme activityfor synthesizing zeaxanthin from β-carotene as a substrate by way ofβ-cryptoxanthin (Example 7 (3); see FIG. 11). Furthermore, the crtZ geneproduct also has an enzyme activity for adding a hydroxyl group to the3-carbon of a 4-keto-β-ionone ring, and one of the specific examples isan enzyme activity for synthesizing astaxanthin from canthaxanthin as asubstrate by way of phoenicoxanthin (Example 6; see FIG. 11). Inaddition, polypeptides having the latter enzyme activity and DNA strandsencoding these polypeptides have not hitherto been known. Also, the CrtZof Agrobacterium showed significant homology to the CrtZ of Erwiniauredovora (identity of 57%) at the level of amino acid sequence.

(3) Identification of a lycopene cyclase gene

Astaxanthin can be synthesized from β-carotene with the 2.9 kb BamHIfragment from which a fragment of the right side from a KpnI site hadbeen removed (pAK96K) or with the 2.9 kb BamHI fragment from which afragment right from the PstI site which is placed further right of theKpnI site had been removed (pAK94) (Example 6), but astaxanthin cannotbe synthesized from lycopene. On the other hand, when a 1.6 kb SalIfragment (pAK98), which contains a right fragment from unique SalI sitepresent further left than the aforementioned KpnI site within the 2.9 kbBamHI fragment, was introduced into lycopene-producing Escherichia coli,β-carotene was synthesized (Example 7 (4)). It is thus considered that agene encoding lycopene cyclase that has an enzyme activity forsynthesizing β-carotene from lycopene as a substrate is present withinthe 1.6 kb SalI fragment of pAK98, and this gene is present over a rangeof the KpnI site and the PstI site. As a result of determining thenucleotide sequence, an open reading frame which corresponds to the geneand has a ribosome binding site just in front of the initiation codonwas successfully detected, it was then referred to as the crtY gene. Thenucleotide sequence of the crtY gene and the amino acid sequence to beencoded are illustrated in FIGS. 3-4 (SEQ ID NO: 3).

The crtY gene product (CrtY) of Agrobacterium aurantiacus sp. nov. MK1has significant homology to the CrtY of Erwinia uredovora (identity of44.3%) at the level of amino acid sequence, and the functions of bothenzymes are the same.

Example 9: Southern Blotting Analysis with the Chromosomal DNA of theOther Marine Bacteria

Examination was conducted whether a region exhibiting homology with theisolated crtW and crtZ is obtained from a chromosomal DNAs of the othermarine microorganisms. The chromosomal DNAs of Alcaligenes sp. PC-1 andAlteromonas sp. SD-402 prepared in Example 1 were digested withrestriction enzymes BamHI and PstI, and separated by agarose gelelectrophoresis. All of the DNA fragments thus separated weredenaturated with an alkali solution of 0.5N NaOH and 1.5M NaCl, andtransferred on a nylon membrane filter over an overnight period. Thenylon membrane filter on which DNAs had been adsorbed was dipped in ahybridization solution (6× Denhardt, 5× SSC, 100 μg/ml ssDNA), andpre-hybridization was conducted at 60° C. for 2 hours. Next, the 1.5 kbDNA fragment cut out from pAK96K with BalI, which contains crtW andcrtY, was labelled with a Mega prime™ DNA labelling systems (Amersham)and α-³² P!dCTP (˜110TBq/mmol) and added to the aforementionedprehybridization solution to conduct hybridization at 60° C. for 16hours.

After hybridization, the filter was washed with 2×SSC containing 0.1%SDS at 60° C. for 1 hour, and subjected to the detection of signalsshowing homology by autoradiography. As a result, strong signals wereobtained at about 13 kb in the product digested with BamHI and at 2.35kb in the product digested with PstI in the case of Alcaligenes sp.PC-1, and strong signals were obtained at about 5.6 kb in the productdigested with BamHI and at 20 kb or more in the product digested withPstI in the case of Alteromonas sp. SD-4.

Example 10: Acquisition of a Xanthophyll Synthesis Gene Cluster from theOther Marine Bacterium

As it was found from the results of Example 9 that the PstI digest ofthe chromosomal DNA of Alcaligenes sp. PC-1 has a region of about 2.35kb hybridizing with a DNA fragment containing the crtW and crtZ genes ofAgrobacterium aurantiacus sp. nov. MK1, the chromosomal DNA ofAlcaligenes was digested with PstI, and then DNA fragments of 2-3.5 kbin size was recovered by agarose gel electrophoresis. The DNA fragmentsthus collected were inserted into the PstI site of a vector pBluescriptII SK+, and introduced into Escherichia coli DH5a to prepare a partiallibrary of Alcaligenes. When the partial library was subjected to colonyhybridization with a 1.5 kb DNA fragment containing the crtW and crtZgenes of Agrobacterium as a probe, a positive colony was isolated fromabout 5,000 colonies. In this case, colony hybridization was conductedunder the same condition as in the Southern blotting analysis shown inExample 9. When plasmid DNA was isolated from the colony thus obtained,and digested with PstI to examine the size of the integrated DNAfragments, it was found that the plasmid contained three differentfragments. Thus, a 2.35 kb fragment to be hybridized was selected fromthe three different DNA fragments by the Southern blotting analysisdescribed in Example 9, the 2.35 kb PstI fragment was recovered byagarose gel electrophoresis and inserted again into the PstI site ofpBluescript II SK+ to prepare the plasmids pPC11 and pPC12. In pPC11 andpPC12, the aforementioned 2.35 kb PstI fragment was inserted into thePstI site of pBluescript II SK+ in an opposite direction to each other.The restriction enzyme map of pPC11 is illustrated in FIG. 19.

Example 11: Determination of Nucleotide Sequence of XanthophyllSynthesis Gene Cluster in Alcaligenes

When each of pPC11 and pPC12 was introduced into β-carotene-producingEscherichia coli, orange colonies were obtained due to the synthesis ofastaxanthin (Example 12) in the former, but no other pigments were newlysynthesized in the latter. It was thus considered that the direction ofthe astaxanthin synthesis gene cluster in the plasmid pPC11 was the sameas that of the vector lac promoter. It was also found that pPC11contained no lycopene cyclizing enzyme genes, since no other pigmentswere newly produced even if pPC11 was introduced into thelycopene-producing Escherichia coli.

It was found that even if a plasmid having a 0.72 kb BstEII-EcoRVfragment positioned at the right side of the PstI fragment had beenremoved (referred to as pPC17, FIG. 19) was introduced into theβ-carotene-producing Escherichia coli, the transformant of Escherichiacoli synthesized astaxanthin and the like (Example 12), same as in thecase of E. coli into which pPC11 was introduced, so that the nucleotidesequence of the 1.63 kb PstI-BstEII fragment in pPC17 was determined.

Deletion mutants were prepared with pPC17 and pPC12 according to thefollowing procedure. A 10 μg portion of each of pPC17 and pPC12 wasdigested with KpnI and HindIII or KpnI and EcoRI, extracted withphenol/chloroform, and DNA was recovered by precipitation with ethanol.Each of DNAs was dissolved in 100 μl of ExoIII buffer (50 mM Tris-HCl,100 mM NaCl, 5 mM MgCl₂, 10 mM 2-mercaptoethanol, pH 8.0), 180 units ofExoIII nuclease was added, and the mixture was maintained at 37° C. A 10μl portion was sampled at every 1 minute, and two samples weretransferred into a tube in which 20 μl of an MB buffer (40 mM sodiumacetate, 100 mM NaCl, 2 mM ZnCl₂, 10% glycerol, pH 4.5) is contained andwhich is placed on ice. After completion of the sampling, five tubesthus obtained were maintained at 65° C. for 10 minutes to inactivate theenzyme, five units of mung bean nuclease were added, and the mixture wasmaintained at 37° C. for 30 minutes. After the reaction, ten DNAfragments different from each other in the degrees of deletion wererecovered for each plasmid by agarose gel electrophoresis. The DNAfragments thus recovered were blunt ended with the Klenow fragment,subjected to the ligation reaction at 16° C. overnight, and Escherichiacoli JM109 was transformed. A single stranded DNA was prepared from eachof various clones thus obtained with a helper phage M13K07, andsubjected to the sequence reaction with a fluorescent primercycle-sequence kit available from Applied Biosystem (K.K.), and the DNAsequence was determined with an automatic sequencer.

The DNA sequence comprising 1631 base pairs (bp) thus obtained isillustrated in FIGS. 16-18 (SEQ ID NO: 12). As a result of examining anopen reading frame having a ribosome binding site in front of theinitiating codon, two open reading frames which can encode thecorresponding proteins (A-B (nucleotide positions 99-824 of SEQ ID NO:7), C-D (nucleotide positions 824-1309) in FIGS. 16-18 were found at thepositions where the two xanthophyll synthesis genes crtW and crtZ wereexpected to be present.

Example 12: Identification of Pigments Produced by Escherichia colihaving an Alcaligenes Xanthophyll Synthesis Gene Cluster

(1) Identification of astaxanthin and 4-ketozeaxanthin

A deletion plasmid (having only crtW) having a deletion from the rightBstEII to the nucleotide position 1162 (FIG. 17) (nucleotide position1162 of SEQ ID NO: 7) among the deletion plasmids from pPC17 prepared inExample 11 was referred to as pPC17-3 (FIG. 19).

The zeaxanthin-producing Escherichia coli JM101 (Example 7 (1)) havingpPC17-3 introduced thereinto (Escherichia coli (pACCAR25ΔcrtX, pPC17-3);exhibiting orange) was cultured in 2 liters of 2YT culture mediumcontaining 150 μg/ml of Ap and 30 μg/ml of Cm at 37° C. for 18 hours.Bacterial cells collected from the culture solution was extracted with300 ml of acetone, concentrated, extracted twice with 200 ml ofchloroform/methanol (9/1), and concentrated to dryness. Then, thin layerchromatography (TLC) was conducted by dissolving the residue in a smallamount of chloroform/methanol (9/1) and developing on a silica gel platefor preparative TLC manufactured by Merck with chloroform/methanol(15/1). The original orange pigment was separated into three spots atthe Rf values of 0.54 (ca. 25%), 0.72 (ca. 30%) and 0.91 (ca. 25%). Thepigments at the Rf values of 0.54 and 0.72 were scratched off from theTLC plate, dissolved in a small amount of chloroform/methanol (9/1) ormethanol, and chromatographed on a Sephadex LH-20 column (15×300 mm)with an eluent of chloroform/methanol (9/1) or methanol to give purifiedmaterials in a yield of about 1 mg, respectively.

The materials were identified as 4-ketozeaxanthin (Rf 0.54) andastaxanthin (Rf 0.72), since all of the data of their UV-visible, FD-MSspectra and mobility in TLC (developed with chloroform/methanol (15/1))accorded with those of the standard samples of 4-ketozeaxanthin andastaxanthin. In addition, the pigment at the Rf value of 0.91 wascanthaxanthin (Example 12 (2)).

It was also confirmed by the similar analytical procedures that theβ-carotene-producing Escherichia coli JM101 having pPC11 or pPC17introduced thereinto (Escherichia coli (pACCAR16ΔcrtX, pPC11 or pPC17)(exhibiting orange) produces astaxanthin, 4-ketozeaxanthin andcanthaxanthin. Furthermore, it was also confirmed with the authenticsample of phoenicoxanthin obtained in Example 6 that these E. colitransformants produce a trace amount of phoenicoxanthin.

(2) Identification of canthaxanthin

The β-carotene-producing Escherichia coli JM101 having pPC17-3introduced thereinto (Escherichia coli (pACCAR16ΔcrtX, pPC17-3);exhibiting orange) was cultured in 2 liters of 2YT culture mediumcontaining 150 μg/ml of Ap and 30 μg/ml of Cm at 37° C. for 18 hours.Bacterial cells collected from the culture solution was extracted with300 ml of acetone, concentrated, extracted twice with 200 ml ofchloroform/methanol (9/1), and concentrated to dryness. Then, thin layerchromatography (TLC) was conducted by dissolving the residue in a smallamount of chloroform/methanol (9/1) and developing on a silica gel platefor preparative TLC manufactured by Merck with chloroform/methanol(50/1). The darkest pigment corresponding to 40% of the total amount oforange pigments was scratched off from the TLC plate, dissolved in asmall amount of chloroform/methanol (9/1) or chloroform/methanol (1/1),and chromatographed on a Sephadex LH-20 column (15×300 mm) with aneluent of chloroform/methanol (9/1) or chloroform/methanol (1/1) to givea purified material in a yield of 2 mg.

The material was identified as canthaxanthin, since all of the data ofits UV-visible, FD-MS (m/e 564) spectra and mobility in TLC (developedwith chloroform/methanol (50/1)) accorded with those of the standardsample of canthaxanthin (manufactured by BASF). In addition, the pigmentof which amount corresponds to 50% of the total amount of the orangepigments observed in the initial extract was considered to be echinenonefrom its UV-visible spectrum, mobility in silica gel TLC (developed withchloroform/methanol (50/1)), and mobility in HPLC with NOVA PACK HR 6μC18 (3.9×300 mm; manufactured by Waters) (developed withacetonitrile/methanol/2-propanol (90/6/4)) (Example 7 (2)). In addition,the balance of the extracted pigments, 10%, was unreacted β-carotene.

(3) Identification of zeaxanthin

A plasmid having a 1.15 kb SalI fragment within pPC11 inserted in thesame direction as the plasmid pPC11 into the SalI site of pBluescript IISK+ was prepared (referred to as pPC13, see FIG. 19).

The β-carotene-producing Escherichia coli JM101 having pPC13 introducedthereinto (Escherichia coli (pACCAR16ΔcrtX, pPC13); exhibiting yellow)was cultured in 2 liters of 2YT culture medium containing 150 μg/ml ofAp and 30 μg/ml of Cm at 37° C. for 18 hours. Bacterial cells collectedfrom the culture solution was extracted with 300 ml of acetone,concentrated, extracted twice with 200 ml of chloroform/methanol (9/1),and concentrated to dryness. Then, thin layer chromatography (TLC) wasconducted by dissolving the residue in a small amount ofchloroform/methanol (9/1) and developing on a silica gel plate forpreparative TLC manufactured by Merck with chloroform/methanol (9/1).The darkest pigment corresponding to 90% of the total amount of orangepigments was scratched off from the TLC plate, dissolved in a smallamount of chloroform/methanol (9/1) or methanol, and chromatographed ona Sephadex LH-20 column (15×300 mm) with an eluent ofchloroform/methanol (9/1) or methanol to give a purified material in ayield of 3 mg.

The material was identified as zeaxanthin, since all of the data of itsUV-visible, FD-MS (m/e 568) spectra and mobility in TLC (developed withchloroform/methanol (9/1)) accorded with those of the standard sample ofzeaxanthin (Example 7 (3)). In addition, the pigment of which amountcorresponds to 10% of the total amount of the orange pigments observedin the initial extract was considered to be β-cryptoxanthin from itsUV-visible spectrum, mobility in silica gel TLC (developed withchloroform/methanol (9/1)), and mobility in HPLC with NOVA PACK HR 6μC18 (3.9×300 mm; manufactured by Waters) (developed withacetonitrile/methanol/2-propanol (90/6/4)) (Example 7 (3)).

Example 13: Identification of the Alcaligenes xanthophyll Synthesis GeneCluster

(1) Identification of a keto group-introducing enzyme gene

It is apparent from the results of Examples 11 and 12 (1) that all ofthe genes required for the synthesis of astaxanthin from β-caroteneamong the 2.35 kb PstI fragment contained in pPC11 is contained in the1.63 kb PstI-BstEII fragment (pPC17, FIG. 19) in the left side. Thus,the 0.72 kb BstEII-PstI fragment in the right side is not needed. UniqueSmaI and SalI sites are present within the 1.63 kb PstI-BstEII fragmentof pPC17 (FIG. 19). It is confirmed by the pigment analysis with aβ-carotene-producing Escherichia coli having the deletion plasmidsintroduced thereinto that the keto group-introducing enzyme activity waslost when the 0.65 kb and 0.69 kb fragments at the left side from SmaIand SalI sites were removed. It was also confirmed by the pigmentanalysis with a β-carotene-producing Escherichia coli having the plasmidintroduced thereinto that the plasmid having a 0.69 kb PstI-SalIfragment positioned at the left side of the 1.63 kb PstI-BstEII fragmentinserted into the PstI-SalI site of pBluescript SK+ has no ketogroup-introducing enzyme activity. On the other hand, the deletionplasmid pPC17-3 (FIG. 19) in which deletion from the BstEII end at theright end to the nucleotide No. 1162 (nucleotide position 1162 in SEQ IDNO: 12) occurred has a keto group-introducing enzyme activity (Example12 (1), (2)), so that it is considered a gene encoding a ketogroup-introducing enzyme having an enzyme activity for synthesizingcanthaxanthin or astaxanthin with a substrate of β-carotene orzeaxanthin is present in the 1162 bp fragment in pPC17-3, and theaforementioned SmaI and SalI sites are present within this gene. As aresult of determining the nucleotide sequence, an open reading framewhich corresponds to the gene and has a ribosome binding site just infront of the initiation codon was successfully detected, so that it wasreferred to as the crtW gene. The nucleotide sequence of the crtW geneand the encoded amino acid sequence are illustrated in FIGS. 13-14 (SEQID NOS: 8-9).

The crtW gene product (CrtW) of Alcaligenes sp. PC-1 has an enzymeactivity for converting a methylene group at the 4-position of aβ-ionone ring into a keto group, and one of the specific examples is anenzyme activity for synthesizing canthaxanthin from β-carotene as asubstrate by way of echinenone (Example 12 (2); see FIG. 11).Furthermore, the crtW gene product also has an enzyme activity forconverting a methylene group at the 4-position of a 3-hydroxy-β-iononering into a keto group, and one of the specific examples is an enzymeactivity for synthesizing astaxanthin from zeaxanthin as a substrate byway of 4-ketozeaxanthin (Example 12 (1); see FIG. 11). In addition,polypeptides having such enzyme activities and DNA strands encodingthese polypeptides have not hitherto been known, and the polypeptidesand the DNA strands encoding these polypeptides have no total homologyto any polypeptides or DNA strands having been hitherto known. Also, thecrtW gene products (CrtW) of Agrobacterium aurantiacus sp. nov. MK1 andAlcaligenes sp. PC-1 share high homology (identity of 83%) at the levelof amino acid sequence, and the functions of both enzymes are the same.The amino acid sequence in the region of 17% having no identity amongthese amino acid sequences is considered not so significant to thefunctions of the enzyme. It is thus considered particularly in thisregion that a little amount of substitution by the other amino acids,deletion, or addition of the other amino acids will not afftect theenzyme activity.

It can be said the keto group-introducing enzyme gene crtW of marinebacteria encodes the β-ionone or 3-hydroxy-β-ionone ring ketolase whichconverts directly the methylene group at the 4-position into a ketogroup irrelative to whether a hydroxyl group is added to the 3-positionor not. In addition, no such informations have hitherto been describedthat a methylene group of not only a β-ionone ring and a3-hydroxy-β-ionone ring but also the other compounds is directlyconverted into a keto group with one enzyme.

(2) Identification of a hydroxyl group-introducing enzyme gene

All of the genes required for the synthesis of astaxanthin fromβ-carotene is contained in the 1.63 kb PstI-BstEII fragment (FIG. 19) ofpPC17. One SalI site is present within the 1.63 kb PstI-BstEII fragmentof pPC17. It is apparent from the results of Example 12 (3) that ahydroxyl group-introducing enzyme activity is present in a fragment atthe right side from the SalI site. It is thus understood that thehydroxyl group-introducing enzyme activity is present in the 0.94 kbSalI-BstEII fragment which is the right fragment in the 1.63 kbPstI-BstEII fragment. As a result of determining the nucleotidesequence, an open reading frame which corresponds to the gene and has aribosome binding site just in front of the initiation codon wassuccessfully detected, it was referred to as the crtZ gene. Thenucleotide sequence of the crtZ gene and the encoded amino acid sequenceare illustrated in FIG. 15 (SEQ ID NOS: 10-11).

The crtZ gene product (CrtZ) of Alcaligenes sp. PC-1 has an enzymeactivity for adding a hydroxyl group to the 3-carbon of a β-ionone ring,and one of the specific examples is an enzyme activity for synthesizingzeaxanthin from β-carotene as a substrate by way of β-cryptoxanthin(Example 12 (3); see FIG. 11). Furthermore, the crtZ gene product alsohas an enzyme activity for adding a hydroxyl group to the 3-carbon of a4-keto-β-ionone ring, and one of the specific examples is an enzymeactivity for synthesizing astaxanthin from canthaxanthin as a substrateby way of phoenicoxanthin (Example 12 (1); see FIG. 11). In addition,polypeptides having the latter enzyme activity and DNA strands encodingthese polypeptides have not hitherto been known. Also, the CrtZ ofAlcaligenes sp. PC-1 showed significant homology to the CrtZ of Erwiniauredovora (identity of 58%) at the level of amino acid sequence. Inaddition, the crtZ gene products (CrtZ) of Agrobacterium aurantiacus sp.nov. MK1 and Alcaligenes sp. PC-1 have high homology (identity of 90%)at the level of amino acid sequence, and the functions of both enzymesare the same. The amino acid sequence in the region of 10% having noidentity among these amino acid sequences is considered not sosignificant to the functions of the enzyme. It is thus consideredparticularly in this region that a little amount of substitution by theother amino acids, deletion, or addition of the other amino acids willnot afftect the enzyme activity.

(3) Consideration on minor biosynthetic pathways of xanthophylls

It has been elucidated by our studies with carotenoid synthesis genes ofthe epiphytic bacterium Erwinia or the photosynthetic bacteriumRhodobacter that carotenoid biosynthesis enzymes generally act byrecognizing the half of a carotenoid molecule as a substrate. By way ofexample, the lycopene cyclase gene of Erwinia, crtY, recognizes thehalves of the lycopene molecule to cyclize it. When the phytoenedesaturase gene crtI of Rhodobacter was used for the synthesis ofneurosporene in place of lycopene in Escherichia coli and crtY ofErwinia was allowed to work on it, the crtY gene product recognizes thehalf molecular structure common to lycopene to produce a half cyclizedβ-zeacarotene (Linden, H., Misawa, N., Chamovits, D., Pecher, I.,Hirschberg, J., Sandmann, G., "Functional Complementation in Escherichiacoli of Different Phytoene Desaturase Genes and Analysis of AccumulatedCarotenes", Z. Naturforsch., 46c, p. 1045-1051, 1991). Also, in thepresent invention, when CrtW is allowed to work on β-carotene orzeaxanthin, echinenone or 4-ketozeaxanthin in which one keto group hasbeen introduced is first synthesized, and when CrtZ is allowed to workon β-carotene or canthaxanthin, β-cryptoxanthin or phoenicoxanthin inwhich one hydroxyl group has been introduced is first synthesized. Itcan be considered because these enzymes recognize the half molecule ofthe substrate. Thus, while Escherichia coli having the crtE, crtB, crtIand crtY genes of Erwinia and the crtZ gene of a marine bacteriumproduces zeaxanthin as described above, β-cryptoxanthin which isβ-carotene having one hydroxyl group introduced thereinto can bedetected as an intermediate metabolite. It can be thus considered thatif CrtW is present, 3'-hydroxyechinenone or 3-hydroxyechinenone can besynthesized from β-cryptoxanthin as a substrate, and thatphoenicoxanthin can be further synthesized by the action of CrtW onthese intermediates. The present inventors have not identified theseketocarotenoids in the culture solutions, and the reason is consideredto be that only a trace amount of these compounds is present under theconditions carried out in the present experiments. In fact, it wasdescribed that 3-hydroxyechinenone or 3'-hydroxyechinenone was detectedas a minor intermediate metabolite of astaxanthin in a marine bacteriumAgrobacterium aurantiacus sp. nov. MK1 as a gene source (AkihiroYokoyama ed., "For the biosynthesis of astaxanthin in marine bacteria",Nippon Suisan Gakkai, Spring Symposium, 1994, Abstract, p. 252, 1994).It can be considered from the above descriptions that minor metabolicpathways shown in FIG. 20 are also present in addition to the mainmetabolic pathways of astaxanthin shown in FIG. 11.

INDUSTRIAL APPLICABILITY

According to the present invention, the gene clusters required for thebiosynthesis of keto group-containing xanthophylls such as astaxanthin,phoenicoxanthin, 4-ketozeaxanthin, canthaxanthin and echinenone havesuccessfully been obtained from marine bacteria, and their structures,nucleotide sequences, and functions have been elucidated. The DNAstrands according to the present invention are useful as genes capableof affording the ability of biosynthesis of keto group-containingxanthophylls such as astaxanthin to microorganisms such as Escherichiacoli and the like.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 12                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A.sub.-- 1 ) LENGTH: 639 base pairs                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..636                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: mat.sub.-- peptide                                              (B) LOCATION: 1..636                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GTGCATGCGCTGTGGTTTCTGGACGCAGCGGCGCATCCCATCCTGGCG48                            ValHisAlaLeuTrpPheLeuAspAlaAlaAlaHisProIleLeuAla                              151015                                                                        ATCGCAAATTTCCTGGGGCTGACCTGGCTGTCGGTCGGATTGTTCATC96                            IleAlaAsnPheLeuGlyLeuThrTrpLeuSerValGlyLeuPheIle                              202530                                                                        ATCGCGCATGACGCGATGCACGGGTCGGTGGTGCCGGGGCGTCCGCGC144                           IleAlaHisAspAlaMetHisGlySerValValProGlyArgProArg                              354045                                                                        GCCAATGCGGCGATGGGCCAGCTTGTCCTGTGGCTGTATGCCGGATTT192                           AlaAsnAlaAlaMetGlyGlnLeuValLeuTrpLeuTyrAlaGlyPhe                              505560                                                                        TCGTGGCGCAAGATGATCGTCAAGCACATGGCCCATCACCGCCATGCC240                           SerTrpArgLysMetIleValLysHisMetAlaHisHisArgHisAla                              65707580                                                                      GGAACCGACGACGACCCCGATTTCGACCATGGCGGCCCGGTCCGCTGG288                           GlyThrAspAspAspProAspPheAspHisGlyGlyProValArgTrp                              859095                                                                        TACGCCCGCTTCATCGGCACCTATTTCGGCTGGCGCGAGGGGCTGCTG336                           TyrAlaArgPheIleGlyThrTyrPheGlyTrpArgGluGlyLeuLeu                              100105110                                                                     CTGCCCGTCATCGTGACGGTCTATGCGCTGATCCTTGGGGATCGCTGG384                           LeuProValIleValThrValTyrAlaLeuIleLeuGlyAspArgTrp                              115120125                                                                     ATGTACGTGGTCTTCTGGCCGCTGCCGTCGATCCTGGCGTCGATCCAG432                           MetTyrValValPheTrpProLeuProSerIleLeuAlaSerIleGln                              130135140                                                                     CTGTTCGTGTTCGGCACCTGGCTGCCGCACCGCCCCGGCCACGACGCG480                           LeuPheValPheGlyThrTrpLeuProHisArgProGlyHisAspAla                              145150155160                                                                  TTCCCGGACCGCCACAATGCGCGGTCGTCGCGGATCAGCGACCCCGTG528                           PheProAspArgHisAsnAlaArgSerSerArgIleSerAspProVal                              165170175                                                                     TCGCTGCTGACCTGCTTTCACTTTGGCGGTTATCATCACGAACACCAC576                           SerLeuLeuThrCysPheHisPheGlyGlyTyrHisHisGluHisHis                              180185190                                                                     CTGCACCCGACGGTGCCGTGGTGGCGCCTGCCCAGCACCCGCACCAAG624                           LeuHisProThrValProTrpTrpArgLeuProSerThrArgThrLys                              195200205                                                                     GGGGACACCGCATGA639                                                            GlyAspThrAla                                                                  210                                                                           (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 212 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       ValHisAlaLeuTrpPheLeuAspAlaAlaAlaHisProIleLeuAla                              151015                                                                        IleAlaAsnPheLeuGlyLeuThrTrpLeuSerValGlyLeuPheIle                              202530                                                                        IleAlaHisAspAlaMetHisGlySerValValProGlyArgProArg                              354045                                                                        AlaAsnAlaAlaMetGlyGlnLeuValLeuTrpLeuTyrAlaGlyPhe                              505560                                                                        SerTrpArgLysMetIleValLysHisMetAlaHisHisArgHisAla                              65707580                                                                      GlyThrAspAspAspProAspPheAspHisGlyGlyProValArgTrp                              859095                                                                        TyrAlaArgPheIleGlyThrTyrPheGlyTrpArgGluGlyLeuLeu                              100105110                                                                     LeuProValIleValThrValTyrAlaLeuIleLeuGlyAspArgTrp                              115120125                                                                     MetTyrValValPheTrpProLeuProSerIleLeuAlaSerIleGln                              130135140                                                                     LeuPheValPheGlyThrTrpLeuProHisArgProGlyHisAspAla                              145150155160                                                                  PheProAspArgHisAsnAlaArgSerSerArgIleSerAspProVal                              165170175                                                                     SerLeuLeuThrCysPheHisPheGlyGlyTyrHisHisGluHisHis                              180185190                                                                     LeuHisProThrValProTrpTrpArgLeuProSerThrArgThrLys                              195200205                                                                     GlyAspThrAla                                                                  210                                                                           (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 489 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..486                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: mat.sub.-- peptide                                              (B) LOCATION: 1..486                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       ATGACCAATTTCCTGATCGTCGTCGCCACCGTGCTGGTGATGGAGTTG48                            MetThrAsnPheLeuIleValValAlaThrValLeuValMetGluLeu                              151015                                                                        ACGGCCTATTCCGTCCACCGCTGGATCATGCACGGCCCCCTGGGCTGG96                            ThrAlaTyrSerValHisArgTrpIleMetHisGlyProLeuGlyTrp                              202530                                                                        GGCTGGCACAAGTCCCACCACGAGGAACACGACCACGCGCTGGAAAAG144                           GlyTrpHisLysSerHisHisGluGluHisAspHisAlaLeuGluLys                              354045                                                                        AACGACCTGTACGGCCTGGTCTTTGCGGTGATCGCCACGGTGCTGTTC192                           AsnAspLeuTyrGlyLeuValPheAlaValIleAlaThrValLeuPhe                              505560                                                                        ACGGTGGGCTGGATCTGGGCGCCGGTCCTGTGGTGGATCGCCTTGGGC240                           ThrValGlyTrpIleTrpAlaProValLeuTrpTrpIleAlaLeuGly                              65707580                                                                      ATGACTGTCTATGGGCTGATCTATTTCGTCCTGCATGACGGGCTGGTG288                           MetThrValTyrGlyLeuIleTyrPheValLeuHisAspGlyLeuVal                              859095                                                                        CATCAGCGCTGGCCGTTCCGTTATATCCCGCGCAAGGGCTATGCCAGA336                           HisGlnArgTrpProPheArgTyrIleProArgLysGlyTyrAlaArg                              100105110                                                                     CGCCTGTATCAGGCCCACCGCCTGCACCATGCGGTCGAGGGGCGCGAC384                           ArgLeuTyrGlnAlaHisArgLeuHisHisAlaValGluGlyArgAsp                              115120125                                                                     CATTGCGTCAGCTTCGGCTTCATCTATGCGCCCCCGGTCGACAAGCTG432                           HisCysValSerPheGlyPheIleTyrAlaProProValAspLysLeu                              130135140                                                                     AAGCAGGACCTGAAGATGTCGGGCGTGCTGCGGGCCGAGGCGCAGGAG480                           LysGlnAspLeuLysMetSerGlyValLeuArgAlaGluAlaGlnGlu                              145150155160                                                                  CGCACGTGA489                                                                  ArgThr                                                                        (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 162 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       MetThrAsnPheLeuIleValValAlaThrValLeuValMetGluLeu                              151015                                                                        ThrAlaTyrSerValHisArgTrpIleMetHisGlyProLeuGlyTrp                              202530                                                                        GlyTrpHisLysSerHisHisGluGluHisAspHisAlaLeuGluLys                              354045                                                                        AsnAspLeuTyrGlyLeuValPheAlaValIleAlaThrValLeuPhe                              505560                                                                        ThrValGlyTrpIleTrpAlaProValLeuTrpTrpIleAlaLeuGly                              65707580                                                                      MetThrValTyrGlyLeuIleTyrPheValLeuHisAspGlyLeuVal                              859095                                                                        HisGlnArgTrpProPheArgTyrIleProArgLysGlyTyrAlaArg                              100105110                                                                     ArgLeuTyrGlnAlaHisArgLeuHisHisAlaValGluGlyArgAsp                              115120125                                                                     HisCysValSerPheGlyPheIleTyrAlaProProValAspLysLeu                              130135140                                                                     LysGlnAspLeuLysMetSerGlyValLeuArgAlaGluAlaGlnGlu                              145150155160                                                                  ArgThr                                                                        (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1161 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..1158                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: mat.sub.-- peptide                                              (B) LOCATION: 1..1158                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GTGACCCATGACGTGCTGCTGGCAGGGGCGGGCCTTGCCAACGGGCTG48                            ValThrHisAspValLeuLeuAlaGlyAlaGlyLeuAlaAsnGlyLeu                              151015                                                                        ATCGCCCTGGCGCTGCGCGCGGCGCGGCCCGACCTGCGCGTGCTGCTG96                            IleAlaLeuAlaLeuArgAlaAlaArgProAspLeuArgValLeuLeu                              202530                                                                        CTGGACCATGCCGCAGGACCGTCAGACGGCCACACCTGGTCCTGCCAC144                           LeuAspHisAlaAlaGlyProSerAspGlyHisThrTrpSerCysHis                              354045                                                                        GACCCCGACCTGTCGCCGGACTGGCTGGCGCGGCTGAAGCCCCTGCGC192                           AspProAspLeuSerProAspTrpLeuAlaArgLeuLysProLeuArg                              505560                                                                        CGCGCCAACTGGCCCGACCAGGAGGTGCGCTTTCCCCGCCATGCCCGG240                           ArgAlaAsnTrpProAspGlnGluValArgPheProArgHisAlaArg                              65707580                                                                      CGGCTGGCCACCGGTTACGGGTCGCTGGACGGGGCGGCGCTGGCGGAT288                           ArgLeuAlaThrGlyTyrGlySerLeuAspGlyAlaAlaLeuAlaAsp                              859095                                                                        GCGGTGGTCCGGTCGGGCGCCGAGATCCGCTGGGACAGCGACATCGCC336                           AlaValValArgSerGlyAlaGluIleArgTrpAspSerAspIleAla                              100105110                                                                     CTGCTGGATGCGCAGGGGGCGACGCTGTCCTGCGGCACCCGGATCGAG384                           LeuLeuAspAlaGlnGlyAlaThrLeuSerCysGlyThrArgIleGlu                              115120125                                                                     GCGGGCGCGGTCCTGGACGGGCGGGGCGCGCAGCCGTCGCGGCATCTG432                           AlaGlyAlaValLeuAspGlyArgGlyAlaGlnProSerArgHisLeu                              130135140                                                                     ACCGTGGGTTTCCAGAAATTCGTGGGTGTCGAGATCGAGACCGACCGC480                           ThrValGlyPheGlnLysPheValGlyValGluIleGluThrAspArg                              145150155160                                                                  CCCCACGGCGTGCCCCGCCCGATGATCATGGACGCGACCGTCACCCAG528                           ProHisGlyValProArgProMetIleMetAspAlaThrValThrGln                              165170175                                                                     CAGGACGGGTACCGCTTCATCTATCTGCTGCCCTTCTCTCCGACGCGC576                           GlnAspGlyTyrArgPheIleTyrLeuLeuProPheSerProThrArg                              180185190                                                                     ATCCTGATCGAGGACACGCGCTATTCCGATGGCGGCGATCTGGACGAC624                           IleLeuIleGluAspThrArgTyrSerAspGlyGlyAspLeuAspAsp                              195200205                                                                     GACGCGCTGGCGGCGGCGTCCCACGACTATGCCCGCCAGCAGGGCTGG672                           AspAlaLeuAlaAlaAlaSerHisAspTyrAlaArgGlnGlnGlyTrp                              210215220                                                                     ACCGGGGCCGAGGTCCGGCGCGAACGCGGCATCCTTCCCATCGCGCTG720                           ThrGlyAlaGluValArgArgGluArgGlyIleLeuProIleAlaLeu                              225230235240                                                                  GCCCATGATGCGGCGGGCTTCTGGGCCGATCACGCGGCGGGGCCTGTT768                           AlaHisAspAlaAlaGlyPheTrpAlaAspHisAlaAlaGlyProVal                              245250255                                                                     CCCGTGGGACTGCGCGCGGGGTTCTTTCATCCGGTCACCGGCTATTCG816                           ProValGlyLeuArgAlaGlyPhePheHisProValThrGlyTyrSer                              260265270                                                                     CTGCCCTATGCGGCACAGGTGGCGGACGTGGTGGCGGGTCTGTCCGGG864                           LeuProTyrAlaAlaGlnValAlaAspValValAlaGlyLeuSerGly                              275280285                                                                     CCGCCCGGCACCGACGCGCTGCGCGGCGCCATCCGCGATTACGCGATC912                           ProProGlyThrAspAlaLeuArgGlyAlaIleArgAspTyrAlaIle                              290295300                                                                     GACCGGGCGCGCCGCGACCGCTTTCTGCGCCTTTTGAACCGGATGCTG960                           AspArgAlaArgArgAspArgPheLeuArgLeuLeuAsnArgMetLeu                              305310315320                                                                  TTCCGCGGCTGCGCGCCCGACCGGCGCTATACCCTGCTGCAGCGGTTC1008                          PheArgGlyCysAlaProAspArgArgTyrThrLeuLeuGlnArgPhe                              325330335                                                                     TACCGCATGCCGCATGGACTGATCGAACGGTTCTATGCCGGCCGGCTG1056                          TyrArgMetProHisGlyLeuIleGluArgPheTyrAlaGlyArgLeu                              340345350                                                                     AGCGTGGCGGATCAGCTGCGCATCGTGACCGGCAAGCCTCCCATTCCC1104                          SerValAlaAspGlnLeuArgIleValThrGlyLysProProIlePro                              355360365                                                                     CTTGGCACGGCCATCCGCTGCCTGCCCGAACGTCCCCTGCTGAAGGAA1152                          LeuGlyThrAlaIleArgCysLeuProGluArgProLeuLeuLysGlu                              370375380                                                                     AACGCATGA1161                                                                 AsnAla                                                                        385                                                                           (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 386 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       ValThrHisAspValLeuLeuAlaGlyAlaGlyLeuAlaAsnGlyLeu                              151015                                                                        IleAlaLeuAlaLeuArgAlaAlaArgProAspLeuArgValLeuLeu                              202530                                                                        LeuAspHisAlaAlaGlyProSerAspGlyHisThrTrpSerCysHis                              354045                                                                        AspProAspLeuSerProAspTrpLeuAlaArgLeuLysProLeuArg                              505560                                                                        ArgAlaAsnTrpProAspGlnGluValArgPheProArgHisAlaArg                              65707580                                                                      ArgLeuAlaThrGlyTyrGlySerLeuAspGlyAlaAlaLeuAlaAsp                              859095                                                                        AlaValValArgSerGlyAlaGluIleArgTrpAspSerAspIleAla                              100105110                                                                     LeuLeuAspAlaGlnGlyAlaThrLeuSerCysGlyThrArgIleGlu                              115120125                                                                     AlaGlyAlaValLeuAspGlyArgGlyAlaGlnProSerArgHisLeu                              130135140                                                                     ThrValGlyPheGlnLysPheValGlyValGluIleGluThrAspArg                              145150155160                                                                  ProHisGlyValProArgProMetIleMetAspAlaThrValThrGln                              165170175                                                                     GlnAspGlyTyrArgPheIleTyrLeuLeuProPheSerProThrArg                              180185190                                                                     IleLeuIleGluAspThrArgTyrSerAspGlyGlyAspLeuAspAsp                              195200205                                                                     AspAlaLeuAlaAlaAlaSerHisAspTyrAlaArgGlnGlnGlyTrp                              210215220                                                                     ThrGlyAlaGluValArgArgGluArgGlyIleLeuProIleAlaLeu                              225230235240                                                                  AlaHisAspAlaAlaGlyPheTrpAlaAspHisAlaAlaGlyProVal                              245250255                                                                     ProValGlyLeuArgAlaGlyPhePheHisProValThrGlyTyrSer                              260265270                                                                     LeuProTyrAlaAlaGlnValAlaAspValValAlaGlyLeuSerGly                              275280285                                                                     ProProGlyThrAspAlaLeuArgGlyAlaIleArgAspTyrAlaIle                              290295300                                                                     AspArgAlaArgArgAspArgPheLeuArgLeuLeuAsnArgMetLeu                              305310315320                                                                  PheArgGlyCysAlaProAspArgArgTyrThrLeuLeuGlnArgPhe                              325330335                                                                     TyrArgMetProHisGlyLeuIleGluArgPheTyrAlaGlyArgLeu                              340345350                                                                     SerValAlaAspGlnLeuArgIleValThrGlyLysProProIlePro                              355360365                                                                     LeuGlyThrAlaIleArgCysLeuProGluArgProLeuLeuLysGlu                              370375380                                                                     AsnAla                                                                        385                                                                           (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2886 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       GGATCCGGCGACCTTGCGGCGCTGCGCCGCGCGCCTTTGCTGGTGCCTGGGCCGGGTGGC60                CAATGGTCGCAAGCAACGGGGATGGAAACCGGCGATGCGGGACTGTAGTCTGCGCGGATC120               GCCGGTCCGGGGGACAAGATGAGCGCACATGCCCTGCCCAAGGCAGATCTGACCGCCACC180               AGCCTGATCGTCTCGGGCGGCATCATCGCCGCTTGGCTGGCCCTGCATGTGCATGCGCTG240               TGGTTTCTGGACGCAGCGGCGCATCCCATCCTGGCGATCGCAAATTTCCTGGGGCTGACC300               TGGCTGTCGGTCGGATTGTTCATCATCGCGCATGACGCGATGCACGGGTCGGTGGTGCCG360               GGGCGTCCGCGCGCCAATGCGGCGATGGGCCAGCTTGTCCTGTGGCTGTATGCCGGATTT420               TCGTGGCGCAAGATGATCGTCAAGCACATGGCCCATCACCGCCATGCCGGAACCGACGAC480               GACCCCGATTTCGACCATGGCGGCCCGGTCCGCTGGTACGCCCGCTTCATCGGCACCTAT540               TTCGGCTGGCGCGAGGGGCTGCTGCTGCCCGTCATCGTGACGGTCTATGCGCTGATCCTT600               GGGGATCGCTGGATGTACGTGGTCTTCTGGCCGCTGCCGTCGATCCTGGCGTCGATCCAG660               CTGTTCGTGTTCGGCACCTGGCTGCCGCACCGCCCCGGCCACGACGCGTTCCCGGACCGC720               CACAATGCGCGGTCGTCGCGGATCAGCGACCCCGTGTCGCTGCTGACCTGCTTTCACTTT780               GGCGGTTATCATCACGAACACCACCTGCACCCGACGGTGCCGTGGTGGCGCCTGCCCAGC840               ACCCGCACCAAGGGGGACACCGCATGACCAATTTCCTGATCGTCGTCGCCACCGTGCTGG900               TGATGGAGTTGACGGCCTATTCCGTCCACCGCTGGATCATGCACGGCCCCCTGGGCTGGG960               GCTGGCACAAGTCCCACCACGAGGAACACGACCACGCGCTGGAAAAGAACGACCTGTACG1020              GCCTGGTCTTTGCGGTGATCGCCACGGTGCTGTTCACGGTGGGCTGGATCTGGGCGCCGG1080              TCCTGTGGTGGATCGCCTTGGGCATGACTGTCTATGGGCTGATCTATTTCGTCCTGCATG1140              ACGGGCTGGTGCATCAGCGCTGGCCGTTCCGTTATATCCCGCGCAAGGGCTATGCCAGAC1200              GCCTGTATCAGGCCCACCGCCTGCACCATGCGGTCGAGGGGCGCGACCATTGCGTCAGCT1260              TCGGCTTCATCTATGCGCCCCCGGTCGACAAGCTGAAGCAGGACCTGAAGATGTCGGGCG1320              TGCTGCGGGCCGAGGCGCAGGAGCGCACGTGACCCATGACGTGCTGCTGGCAGGGGCGGG1380              CCTTGCCAACGGGCTGATCGCCCTGGCGCTGCGCGCGGCGCGGCCCGACCTGCGCGTGCT1440              GCTGCTGGACCATGCCGCAGGACCGTCAGACGGCCACACCTGGTCCTGCCACGACCCCGA1500              CCTGTCGCCGGACTGGCTGGCGCGGCTGAAGCCCCTGCGCCGCGCCAACTGGCCCGACCA1560              GGAGGTGCGCTTTCCCCGCCATGCCCGGCGGCTGGCCACCGGTTACGGGTCGCTGGACGG1620              GGCGGCGCTGGCGGATGCGGTGGTCCGGTCGGGCGCCGAGATCCGCTGGGACAGCGACAT1680              CGCCCTGCTGGATGCGCAGGGGGCGACGCTGTCCTGCGGCACCCGGATCGAGGCGGGCGC1740              GGTCCTGGACGGGCGGGGCGCGCAGCCGTCGCGGCATCTGACCGTGGGTTTCCAGAAATT1800              CGTGGGTGTCGAGATCGAGACCGACCGCCCCCACGGCGTGCCCCGCCCGATGATCATGGA1860              CGCGACCGTCACCCAGCAGGACGGGTACCGCTTCATCTATCTGCTGCCCTTCTCTCCGAC1920              GCGCATCCTGATCGAGGACACGCGCTATTCCGATGGCGGCGATCTGGACGACGACGCGCT1980              GGCGGCGGCGTCCCACGACTATGCCCGCCAGCAGGGCTGGACCGGGGCCGAGGTCCGGCG2040              CGAACGCGGCATCCTTCCCATCGCGCTGGCCCATGATGCGGCGGGCTTCTGGGCCGATCA2100              CGCGGCGGGGCCTGTTCCCGTGGGACTGCGCGCGGGGTTCTTTCATCCGGTCACCGGCTA2160              TTCGCTGCCCTATGCGGCACAGGTGGCGGACGTGGTGGCGGGTCTGTCCGGGCCGCCCGG2220              CACCGACGCGCTGCGCGGCGCCATCCGCGATTACGCGATCGACCGGGCGCGCCGCGACCG2280              CTTTCTGCGCCTTTTGAACCGGATGCTGTTCCGCGGCTGCGCGCCCGACCGGCGCTATAC2340              CCTGCTGCAGCGGTTCTACCGCATGCCGCATGGACTGATCGAACGGTTCTATGCCGGCCG2400              GCTGAGCGTGGCGGATCAGCTGCGCATCGTGACCGGCAAGCCTCCCATTCCCCTTGGCAC2460              GGCCATCCGCTGCCTGCCCGAACGTCCCCTGCTGAAGGAAAACGCATGAACGCCCATTCG2520              CCCGCGGCCAAGACCGCCATCGTGATCGGCGCAGGCTTTGGCGGGCTGGCCCTGGCCATC2580              CGCCTGCAGTCCGCGGGCATCGCCACCACCCTGGTCGAGGCCCGGGACAAGCCCGGCGGG2640              CGCGCCTATGTCTGGCACGATCAGGGCCATCTCTTCGACGCGGGCCCGACCGTCATCACC2700              GACCCCGATGCGCTGAAAGAGCTGTGGGCCCTGACCGGGCAGGACATGGCGCGCGACGTG2760              ACGCTGATGCCGGTCTCGCCCTTCTATCGGCTGATGTGGCCGGGCGGGAAGGTCTTCGAT2820              TACGTGAACGAGGCCGATCCAGGGTCTGGGTCTTGCCGTGCCAGGTGAAGCTGTTGCCGT2880              GGATCC2886                                                                    (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 729 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..726                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: mat.sub.-- peptide                                              (B) LOCATION: 1..726                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       ATGTCCGGACGGAAGCCTGGCACAACTGGCGACACGATCGTCAATCTC48                            MetSerGlyArgLysProGlyThrThrGlyAspThrIleValAsnLeu                              151015                                                                        GGTCTGACCGCCGCGATCCTGCTGTGCTGGCTGGTCCTGCACGCCTTT96                            GlyLeuThrAlaAlaIleLeuLeuCysTrpLeuValLeuHisAlaPhe                              202530                                                                        ACGCTATGGTTGCTAGATGCGGCCGCGCATCCGCTGCTTGCCGTGCTG144                           ThrLeuTrpLeuLeuAspAlaAlaAlaHisProLeuLeuAlaValLeu                              354045                                                                        TGCCTGGCTGGGCTGACCTGGCTGTCGGTCGGGCTGTTCATCATCGCG192                           CysLeuAlaGlyLeuThrTrpLeuSerValGlyLeuPheIleIleAla                              505560                                                                        CATGACGCAATGCACGGGTCCGTGGTGCCGGGGCGGCCGCGCGCCAAT240                           HisAspAlaMetHisGlySerValValProGlyArgProArgAlaAsn                              65707580                                                                      GCGGCGATCGGGCAACTGGCGCTGTGGCTCTATGCGGGGTTCTCGTGG288                           AlaAlaIleGlyGlnLeuAlaLeuTrpLeuTyrAlaGlyPheSerTrp                              859095                                                                        CCCAAGCTGATCGCCAAGCACATGACGCATCACCGGCACGCCGGCACC336                           ProLysLeuIleAlaLysHisMetThrHisHisArgHisAlaGlyThr                              100105110                                                                     GACAACGATCCCGATTTCGGTCACGGAGGGCCCGTGCGCTGGTACGGC384                           AspAsnAspProAspPheGlyHisGlyGlyProValArgTrpTyrGly                              115120125                                                                     AGCTTCGTCTCCACCTATTTCGGCTGGCGAGAGGGACTGCTGCTACCG432                           SerPheValSerThrTyrPheGlyTrpArgGluGlyLeuLeuLeuPro                              130135140                                                                     GTGATCGTCACCACCTATGCGCTGATCCTGGGCGATCGCTGGATGTAT480                           ValIleValThrThrTyrAlaLeuIleLeuGlyAspArgTrpMetTyr                              145150155160                                                                  GTCATCTTCTGGCCGGTCCCGGCCGTTCTGGCGTCGATCCAGATTTTC528                           ValIlePheTrpProValProAlaValLeuAlaSerIleGlnIlePhe                              165170175                                                                     GTCTTCGGAACTTGGCTGCCCCACCGCCCGGGACATGACGATTTTCCC576                           ValPheGlyThrTrpLeuProHisArgProGlyHisAspAspPhePro                              180185190                                                                     GACCGGCACAACGCGAGGTCGACCGGCATCGGCGACCCGTTGTCACTA624                           AspArgHisAsnAlaArgSerThrGlyIleGlyAspProLeuSerLeu                              195200205                                                                     CTGACCTGCTTCCATTTCGGCGGCTATCACCACGAACATCACCTGCAT672                           LeuThrCysPheHisPheGlyGlyTyrHisHisGluHisHisLeuHis                              210215220                                                                     CCGCATGTGCCGTGGTGGCGCCTGCCTCGTACACGCAAGACCGGAGGC720                           ProHisValProTrpTrpArgLeuProArgThrArgLysThrGlyGly                              225230235240                                                                  CGCGCATGA729                                                                  ArgAla                                                                        (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 242 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       MetSerGlyArgLysProGlyThrThrGlyAspThrIleValAsnLeu                              151015                                                                        GlyLeuThrAlaAlaIleLeuLeuCysTrpLeuValLeuHisAlaPhe                              202530                                                                        ThrLeuTrpLeuLeuAspAlaAlaAlaHisProLeuLeuAlaValLeu                              354045                                                                        CysLeuAlaGlyLeuThrTrpLeuSerValGlyLeuPheIleIleAla                              505560                                                                        HisAspAlaMetHisGlySerValValProGlyArgProArgAlaAsn                              65707580                                                                      AlaAlaIleGlyGlnLeuAlaLeuTrpLeuTyrAlaGlyPheSerTrp                              859095                                                                        ProLysLeuIleAlaLysHisMetThrHisHisArgHisAlaGlyThr                              100105110                                                                     AspAsnAspProAspPheGlyHisGlyGlyProValArgTrpTyrGly                              115120125                                                                     SerPheValSerThrTyrPheGlyTrpArgGluGlyLeuLeuLeuPro                              130135140                                                                     ValIleValThrThrTyrAlaLeuIleLeuGlyAspArgTrpMetTyr                              145150155160                                                                  ValIlePheTrpProValProAlaValLeuAlaSerIleGlnIlePhe                              165170175                                                                     ValPheGlyThrTrpLeuProHisArgProGlyHisAspAspPhePro                              180185190                                                                     AspArgHisAsnAlaArgSerThrGlyIleGlyAspProLeuSerLeu                              195200205                                                                     LeuThrCysPheHisPheGlyGlyTyrHisHisGluHisHisLeuHis                              210215220                                                                     ProHisValProTrpTrpArgLeuProArgThrArgLysThrGlyGly                              225230235240                                                                  ArgAla                                                                        (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 489 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..486                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: mat.sub.-- peptide                                              (B) LOCATION: 1..486                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      ATGACGCAATTCCTCATTGTCGTGGCGACAGTCCTCGTGATGGAGCTG48                            MetThrGlnPheLeuIleValValAlaThrValLeuValMetGluLeu                              151015                                                                        ACCGCCTATTCCGTCCACCGCTGGATTATGCACGGCCCCCTAGGCTGG96                            ThrAlaTyrSerValHisArgTrpIleMetHisGlyProLeuGlyTrp                              202530                                                                        GGCTGGCACAAGTCCCATCACGAAGAGCACGACCACGCGTTGGAGAAG144                           GlyTrpHisLysSerHisHisGluGluHisAspHisAlaLeuGluLys                              354045                                                                        AACGACCTCTACGGCGTCGTCTTCGCGGTGCTGGCGACGATCCTCTTC192                           AsnAspLeuTyrGlyValValPheAlaValLeuAlaThrIleLeuPhe                              505560                                                                        ACCGTGGGCGCCTATTGGTGGCCGGTGCTGTGGTGGATCGCCCTGGGC240                           ThrValGlyAlaTyrTrpTrpProValLeuTrpTrpIleAlaLeuGly                              65707580                                                                      ATGACGGTCTATGGGTTGATCTATTTCATCCTGCACGACGGGCTTGTG288                           MetThrValTyrGlyLeuIleTyrPheIleLeuHisAspGlyLeuVal                              859095                                                                        CATCAACGCTGGCCGTTTCGGTATATTCCGCGGCGGGGCTATTTCCGC336                           HisGlnArgTrpProPheArgTyrIleProArgArgGlyTyrPheArg                              100105110                                                                     AGGCTCTACCAAGCTCATCGCCTGCACCACGCGGTCGAGGGGCGGGAC384                           ArgLeuTyrGlnAlaHisArgLeuHisHisAlaValGluGlyArgAsp                              115120125                                                                     CACTGCGTCAGCTTCGGCTTCATCTATGCCCCACCCGTGGACAAGCTG432                           HisCysValSerPheGlyPheIleTyrAlaProProValAspLysLeu                              130135140                                                                     AAGCAGGATCTGAAGCGGTCGGGTGTCCTGCGCCCCCAGGACGAGCGT480                           LysGlnAspLeuLysArgSerGlyValLeuArgProGlnAspGluArg                              145150155160                                                                  CCGTCGTGA489                                                                  ProSer                                                                        (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 162 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      MetThrGlnPheLeuIleValValAlaThrValLeuValMetGluLeu                              151015                                                                        ThrAlaTyrSerValHisArgTrpIleMetHisGlyProLeuGlyTrp                              202530                                                                        GlyTrpHisLysSerHisHisGluGluHisAspHisAlaLeuGluLys                              354045                                                                        AsnAspLeuTyrGlyValValPheAlaValLeuAlaThrIleLeuPhe                              505560                                                                        ThrValGlyAlaTyrTrpTrpProValLeuTrpTrpIleAlaLeuGly                              65707580                                                                      MetThrValTyrGlyLeuIleTyrPheIleLeuHisAspGlyLeuVal                              859095                                                                        HisGlnArgTrpProPheArgTyrIleProArgArgGlyTyrPheArg                              100105110                                                                     ArgLeuTyrGlnAlaHisArgLeuHisHisAlaValGluGlyArgAsp                              115120125                                                                     HisCysValSerPheGlyPheIleTyrAlaProProValAspLysLeu                              130135140                                                                     LysGlnAspLeuLysArgSerGlyValLeuArgProGlnAspGluArg                              145150155160                                                                  ProSer                                                                        (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1631 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      CTGCAGGCCGGGCCCGGTGGCCAATGGTCGCAACCGGCAGGACTGGAACAGGACGGCGGG60                CCGGTCTAGGCTGTCGCCCTACGCAGCAGGAGTTTCGGATGTCCGGACGGAAGCCTGGCA120               CAACTGGCGACACGATCGTCAATCTCGGTCTGACCGCCGCGATCCTGCTGTGCTGGCTGG180               TCCTGCACGCCTTTACGCTATGGTTGCTAGATGCGGCCGCGCATCCGCTGCTTGCCGTGC240               TGTGCCTGGCTGGGCTGACCTGGCTGTCGGTCGGGCTGTTCATCATCGCGCATGACGCAA300               TGCACGGGTCCGTGGTGCCGGGGCGGCCGCGCGCCAATGCGGCGATCGGGCAACTGGCGC360               TGTGGCTCTATGCGGGGTTCTCGTGGCCCAAGCTGATCGCCAAGCACATGACGCATCACC420               GGCACGCCGGCACCGACAACGATCCCGATTTCGGTCACGGAGGGCCCGTGCGCTGGTACG480               GCAGCTTCGTCTCCACCTATTTCGGCTGGCGAGAGGGACTGCTGCTACCGGTGATCGTCA540               CCACCTATGCGCTGATCCTGGGCGATCGCTGGATGTATGTCATCTTCTGGCCGGTCCCGG600               CCGTTCTGGCGTCGATCCAGATTTTCGTCTTCGGAACTTGGCTGCCCCACCGCCCGGGAC660               ATGACGATTTTCCCGACCGGCACAACGCGAGGTCGACCGGCATCGGCGACCCGTTGTCAC720               TACTGACCTGCTTCCATTTCGGCGGCTATCACCACGAACATCACCTGCATCCGCATGTGC780               CGTGGTGGCGCCTGCCTCGTACACGCAAGACCGGAGGCCGCGCATGACGCAATTCCTCAT840               TGTCGTGGCGACAGTCCTCGTGATGGAGCTGACCGCCTATTCCGTCCACCGCTGGATTAT900               GCACGGCCCCCTAGGCTGGGGCTGGCACAAGTCCCATCACGAAGAGCACGACCACGCGTT960               GGAGAAGAACGACCTCTACGGCGTCGTCTTCGCGGTGCTGGCGACGATCCTCTTCACCGT1020              GGGCGCCTATTGGTGGCCGGTGCTGTGGTGGATCGCCCTGGGCATGACGGTCTATGGGTT1080              GATCTATTTCATCCTGCACGACGGGCTTGTGCATCAACGCTGGCCGTTTCGGTATATTCC1140              GCGGCGGGGCTATTTCCGCAGGCTCTACCAAGCTCATCGCCTGCACCACGCGGTCGAGGG1200              GCGGGACCACTGCGTCAGCTTCGGCTTCATCTATGCCCCACCCGTGGACAAGCTGAAGCA1260              GGATCTGAAGCGGTCGGGTGTCCTGCGCCCCCAGGACGAGCGTCCGTCGTGATCTCTGAT1320              CCCGGCGTGGCCGCATGAAATCCGACGTGCTGCTGGCAGGGGCCGGCCTTGCCAACGGAC1380              TGATCGCGCTGGCGATCCGCAAGGCGCGGCCCGACCTTCGCGTGCTGCTGCTGGACCGTG1440              CGGCGGGCGCCTCGGACGGGCATACTTGGTCCTGCCACGACACCGATTTGGCGCCGCACT1500              GGCTGGACCGCCTGAAGCCGATCAGGCGTGGCGACTGGCCCGATCAGGAGGTGCGGTTCC1560              CAGACCATTCGCGAAGGCTCCGGGCCGGATATGGCTCGATCGACGGGCGGGGGCTGATGC1620              GTGCGGTGACC1631                                                               __________________________________________________________________________

We claim:
 1. A process for producing xanthophyll, comprising introducinga DNA strand into a microorganism having a canthaxanthin-synthesizingability, wherein said DNA strand has a nucleotide sequence which encodesa polypeptide having an enzyme activity for adding a hydroxyl group tothe 3-carbon of a 4-keto-β-ionone ring, culturing the transformedmicroorganism in a culture medium, and obtaining astaxanthin orphoenicoxanthin from the cultured cells.
 2. A process according to claim1, wherein said microorganism is a bacterium or yeast.
 3. A processaccording to claim 1, wherein said DNA strand has a sequence that codesfor a polypeptide having the sequence of SEQ ID NO:4.
 4. A process forproducing xanthophyll, comprising providing a transformed microorganismwherein said transformed microorganism contains DNA that codes apolypeptide having an enzymatic activity for converting β-carotene tocanthaxanthin and that codes a polypeptide having an enzyme activity foradding a hydroxyl group to the 3-carbon of a 4-keto-β-ionone ring,culturing the transformed microorganism in a culture medium, andobtaining astaxanthin or phoenicoxanthin from the cultured cells.
 5. Aprocess according to claim 4, wherein said microorganism is a bacterium.6. A process according to claim 5, wherein said bacterium is selectedfrom the group consisting of Escherichia coli, Zymomonas mobilis andAgrobacterium tumefaciens.
 7. A process for producing xanthophyll,comprising introducing DNA that contains the crtW and crtZ genesisolated from Agrobacterium aurantiacus into a microorganism, culturingthe transformed microorganism in a culture medium, and obtainingastaxanthin or phoenicoxanthin from the cultured cells.